Cell Membrane Translocation of Regulated Snare Inhibitors, Compositions Therefor, and Methods for Treatment of Disease

ABSTRACT

Compositions and methods of modulating cellular function and treatment of disease in mammals comprising locally administering a regulated SNARE inhibitor and a translocating agent to the mammal. Regulated SNARE inhibitors include clostridial neurotoxins, tetanus neurotoxin and their free light chain portions and IgA protease. Translocating agents include acids, encapsulating vectors, and transduction domains.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/545,872 (pending), filed Aug. 17, 2005, entitled Cell MembraneTranslocation of Regulated Snare Inhibitors, Compositions Therefor, andMethods for Treatment of Disease, which is a U.S. national phase entryunder 35 U.S.C. §371 of International Application No. PCT/US04/05436(expired), filed Feb. 24, 2004, entitled Cell Membrane Translocation ofRegulated Snare Inhibitors, Compositions Therefor, and Methods forTreatment of Disease, which claims the benefit of U.S. ProvisionalApplication No. 60/449,107 (expired), filed Feb. 24, 2003, entitledCompositions of Amphipathic Pharmaceuticals and Methods For Their Use,by I. Sanders, each of which applications are hereby incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

This invention relates to translocation of bioactive molecules throughcell membranes. More specifically, the invention relates to cellmembrane translocation of regulated SNARE inhibitors, compositions andformulations thereof, and methods for treatment of disease.

BACKGROUND OF THE INVENTION

A promising area of pharmaceutical intervention is the use ofmacromolecules that act within cells. These include gene therapy,natural and recombinant toxins, immunotoxins, and antibodies. Onesignificant technical barrier is passing the bioactive macromoleculesfrom the extracellular fluid (the cis-side of the membrane) through thebilipid cell membrane to the cytoplasm (the trans-side).

Generally, macromolecules enter the cell by endocytosis. Endocytosis isan ongoing process whereby the cell recycles its membrane components andinternalizes molecules bound to its surface. During endocytosis, thecell membrane invaginates into the cell's interior and then pinches offto form an endosome. Endosomes comprise a complete membrane thatencloses its internal contents (the cis-side) and separates them fromthe cytoplasm of the cell (the trans-side). After endosome formation,proton pumps within its membrane act to reduce the pH within theendosome to approximately 5.0 to 5.5. At a later stage, many endosomesmerge with lysosomes where their contents are degraded.

In one mechanism of endocytosis-type cell-membrane transport, the toxinsremain in endosomes that undergo processing and ultimately merges withthe endoplasmic reticulum of the cell. In the endoplasmic reticulumthere is a membrane transporter that translocates the molecule into thecytoplasm. This route is used by cholera toxin and the toxin ricin. Thisroute is difficult for most complex pharmaceuticals as they are degradedduring the prolonged endosomal stage.

In a second, and probably the most common, method by which toxins,viruses and pharmaceuticals with amphipathic regions (i.e., regions thatcontain both hydrophobic and hydrophilic amino acids) enter cells, thetoxin is internalized in the endosome as described above and then passesthrough the endosome membrane when the interior pH becomes acidic. Atacidic pH, these amphipathic regions become more hydrophobic and mergewith the endosome membrane. Once incorporated in the membrane, a pore isformed through which the toxic or catalytic part of the molecule passesfrom the endosome into the cytoplasm.

The amphipathic bacterial toxins include the clostridial neurotoxinsfrom Clostridia botulinum, berati, butyricum and tetani, Clostridiabotulinum toxin C2, Clostridia perfringens iota toxin, Clostridiadifficile B toxin, anthrax toxin, diptheria toxin, and others. All ofthese toxins have a basic two-component protein structure: (1) a toxicprotein chain containing a catalytic domain that can performintracellular intoxication; and (2) a protein with both a binding andtranslocation domain. In most toxins, the two protein components arelinked covalently or tightly coupled by non-covalent forces. In others,such as Botulinum toxin C2, the two components are independent proteins,and they only interact on cell surfaces. Anthrax toxin is unusual inthat the same binding/translocation protein chain, called protectiveantigen, can translocate either of two independent toxic proteins,lethal factor and edema factor. The binding/translocation protein chainis further subdivided into: (1) a binding domain, which recognizes oneor more receptors on the surface of cells; and (2) an amphipathicdomain, which can translocate the molecule through cellular or endosomemembranes at acidic pH.

The binding/translocation protein chains from these wild type toxinshave been separated and used as carriers to bring molecular ‘cargo’ intocells: other biological useful molecules (e.g., tetanusneurotoxin/superoxide dismutase WO/0028041A1: Delivery Of SuperoxideDismutase To Neuronal Cells, hereby incorporated herein by reference).Toxins used in this manner include botulinum and tetanus toxins,diptheria toxin, botulinum toxin C2, and anthrax toxin. The amphipathicregions have been sequenced for the translocation domains of manybacterial toxins and viruses, and based on this knowledge, novelrecombinant amphipathic moieties have been produced. At present some ofthese natural and recombinant amphipathic proteins have been shown tocapable of translocating across the endosome membrane when the endosomeinterior becomes acidic. However, few amphipathic protein conjugates arepresently approved for clinical use.

1. Botulinum Neurotoxin

Wild type clostridial neurotoxins, specifically those from Clostridiabotulinum, are amphipathic protein conjugates with unique propertiesthat make them beneficial in medical applications. First, in theirnatural or wild type form, they have specificity for neurons,particularly motor neurons. Second, they can block neuromusculartransmission for extended periods, from days to months depending on theserotype. Third, in most clinical applications they have been used atdoses that are below the level of immunological recognition. Fourth, asthey are remarkably safe for human use when injected into local areassuch as muscles because there is little systemic spread of the toxin.

The clostridial neurotoxins include seven serotypes of botulinumneurotoxins, termed A-G (A, B, C1, D, E, F, and G), and a singleserotype of tetanus toxin (tetanus neurotoxin). These toxins all have amolecule size of ˜150 kD and are comprised of a heavy-chain (˜100 kD)and a light chain (˜50 kD) that are covalently linked by a disulphidebridge at their N-terminals. The heavy chain consists of the bindingdomain (fragment C) at the C terminal and a translocation domain(fragment B, which is the amphipathic protein) at the N-terminal end.The light chain (fragment A) is the toxic domain, however, it alsocontains its own small amphipathic region. These neurotoxins areexceptional due to their specific binding to neurons and their specificcatalytic action on the SNARE proteins, which are involved inneurotransmission. Botulinum neurotoxins A, C, and E cleave SNAP-25, inaddition botulinum neurotoxin/C cleaves syntaxin 1. botulinumneurotoxins B, D, F, G and tetanus toxin cleave VAMP-2.

The C fragments of the clostridial neurotoxins have affinity for thepresynaptic membrane of neurons, and particularly the membrane of motorneurons. Clostridial neurotoxin binding is believed to involve tworeceptors: (1) polysialo-gangliosides accumulate clostridial neurotoxinson the plasma membrane surface, and (2) protein receptors then mediatespecific endocytosis. This hypothesis was supported by the demonstrationof the binding of botulinum neurotoxins B to the neuronal membraneprotein synaptotagmin in the presence of GT1b, and the recentidentification of GPI-anchored glycoproteins in neuronal rafts asspecific receptors for the HC-fragment of tetanus neurotoxin.

After a clostridial neurotoxin binds to the presynaptic surface, it isinternalized by incorporation into endosomes. When the interior of theendosome reaches about pH 5.5, the amphipathic B-fragment merges withthe membrane and forms a pore that allows the light chain to passthrough to the cell's cytoplasm. While passing through the membrane thedisulfide bond is broken and the light chain is released into thecytoplasm and exerts its toxic effect.

The toxic action of all clostridial neurotoxin light chains is to cleaveproteins necessary for attachment of internal vesicles to the cellmembrane. The production and docking of these vesicles is a highlyregulated process that is present in all eukaryotic cells includingsingle-cell organisms such as yeast. The vesicle membranes merge withthe cell membrane thereby adding new membrane bound proteins whilesimultaneously discharging the vesicle's contents into the extracellularenvironment. In neurons, these vesicles contain neurotransmitters andneuropeptides. Botulinum neurotoxin A and E cleaves SNAP-25; botulinumneurotoxin C cleaves SNAP-25 and syntaxin 1; and tetanus neurotoxin andbotulinum neurotoxin types B, D, F and G cleave VAMP (vesicle associatedmembrane protein, also called synaptobrevin).

Botulinum neurotoxins A and B are the serotypes currently approved bythe FDA for human use. Direct injection into extra-ocular muscles wasfound to be beneficial in the treatment of strabismus. Subsequently,botulinum neurotoxin A has been used to treat a variety of spastic orhyper-functional muscle disorders. Botulinum neurotoxin A has also beenused for the treatment of smooth muscle hyper-function (e.g.,cricopharyngeal spasm). Recently, botulinum neurotoxin has been used fortreatment in connection with the cholinergic nerves of the autonomicnervous system. These uses include arresting of secretions, such assweating and post-nasal drip.

2. Tetanus Neurotoxin

Tetanus neurotoxin exhibits fundamental differences relative tobotulinum neurotoxin. First, tetanus neurotoxin binds and enters intoall peripheral neurons: motor, autonomic (parasympathetic andsympathetic) and sensory neurons, including those that transmit painsignals. In contrast, botulinum neurotoxin binds and enters only motorneurons and autonomic parasympathetic neurons.

Second, at physiological doses, in contrast to botulinum neurotoxin,tetanus neurotoxin does not use the acidified endosomal pathway to enterperipheral neurons. Although internalized in the same manner asbotulinum neurotoxin, the specific receptors to which tetanus neurotoxinbinds allows for preferential sorting of the endosome.Tetanus-neurotoxin-containing endosomes become non-acidified vesiclesthat are transported retrograde to the motor neuron cell body in thecentral nervous system or sensory ganglia. Upon reaching the cell body,tetanus neurotoxin is released into the presynaptic space andpreferentially binds to inhibitory neurons that use glycine or GABA astheir neurotransmitter. When tetanus neurotoxin is taken up byinhibitory neurons in the central nervous system, it then goes throughthe acidified endosomal stage and acts much like that of botulinumneurotoxin in peripheral neurons. Accordingly, tetanus neurotoxinpreferentially blocks inhibitory activity. The resulting unopposedexcitatory activity causes muscles to contract uncontrollably, acondition called spastic paralysis. Although the clinical conditionknown as tetanus is a systemic intoxication, it is known that tetanusneurotoxin can also act in localized areas in mammals. At dosescomparable to those that cause paralysis with botulinum neurotoxin A,tetanus neurotoxin causes a local increase in motor, autonomic and/orsensory neuron activity.

At high doses tetanus neurotoxin can cause paralysis by blockingneurotransmission both centrally and peripherally. At doses, 10 to 2000times that needed for excitation, tetanus neurotoxin blocks bothexcitatory and inhibitory neurons in the central nervous system. Thesehigh doses risk local and systemic side effects. In addition, thebinding domain of tetanus neurotoxin can be separated from the remainderof the molecule by digestion with the enzyme papain. Upon digestion, theresulting fragment is called tetanus neurotoxin A-B fragment andcontains the light chain connected by a disulphide bridge to thetranslocating domain of the heavy chain. Since the A-B fragment ismissing its binding fragment, it can no longer both bind and undergoretrograde transport. But the A-B fragment can cross the cell membraneand paralyze the neuromuscular synapse and cause a flaccid paralysis.But this effect requires tens of thousands more molecules of A-Bfragment to than that needed for the excitation caused by wild typetetanus neurotoxin. The mechanism for this effect seems to thenon-specific pinocytosis of tetanus neurotoxin A-B fragment by cells.

Finally, another unusual attribute of tetanus neurotoxin that it isinternalized by some non-neuronal cell types. The most clinically usefulof these are the macrophages that migrate to areas of inflammation.Tetanus neurotoxin blocks the release of inflammatory mediators andenzymes by macrophages, thereby decreasing the inflammatory response.

International Application WO 02/00172 (published Jan. 3, 2002), herebyincorporated herein by reference, teaches a wide variety of methods forusing tetanus neurotoxin by increasing or decreasing neural activity ornon-neural cellular activity.

The wild type amphipathic protein conjugates such as clostridialneurotoxin conjugates have wide potential as therapeutic agents. Forexample, the selective motor neuron binding of the neurotoxin heavychain has been combined with the enzyme superoxide dismutase for thetreatment of motor neuron degenerative diseases. The CNS transportabilities of the tetanus neurotoxin heavy chain or its Hc fragment areespecially useful as it is one of the few vectors that can bypass theblood brain barrier (rabies and herpes virus being two others). Due tothe universal nature of the vesicle docking process in cells, the use ofclostridial neurotoxin light chains combined with cell-type specificamphipathic proteins holds great promise for the treatment of a widevariety of clinical conditions. Very specific targeting of cell types isplausible using recombinant technology to incorporate monoclonalimmunoglobulins into amphipathic proteins. Unfortunately, however, asdiscussed below, introduction of these novel compounds is limitedbecause of the inefficiencies of the endosomic process.

3. Disadvantages of Endosomic Transport of Amphipathic Protein Conjugateinto Cells

Although, as discussed above, amphipathic protein conjugates—such asclostridial neurotoxins—have potential medical applications, theirusefulness is limited by inefficient transport into cells. As discussedabove, amphipathic protein conjugates enter cells by way of endocytosisand then require translocation across the endosome membrane. This is adisadvantage for a variety of reasons. In most cases, only a smallpercentage of the active moieties survive the various steps of cellbinding, endocytosis, endosome acidification, and translocation. Thisinefficiency increases the incidence of side effects and the inductionof immune reactions.

Binding of some amphipathic protein conjugates to cell membranes ishighly specific to the cell type. This is an advantage for someconditions but precludes their use in other conditions where bindingaffinity is low. Further, the binding of the amphipathic proteinconjugates to the surface of membranes prior to endocytosis can beprolonged as they await the normal cell turnover of cell membrane toreach them. Therefore, these molecules are exposed to degradingextracellular enzymes, and in some cases neutralizing antibodies. Onceinternalized into the cell within an endosome, acidification of theendosome to induce release of the active moiety into the cell can takehours. Accordingly, translocation across the endosome membrane is therate-limiting step in the entry of the active moiety into the cell.Another disadvantage of amphipathic protein conjugates, is that theendosome contains its own assortment of enzymes that can degrade theconjugate. For example, during the treatment of cancer, the developmentof multi-drug resistance by cancer cells is believed to involvemolecular changes in the endosome that cause the drug to be removed fromthe cell.

Therefore there is a need in the art for a method allowing amphipathicprotein conjugates to bypass the endosomal stage and translocatedirectly across cell membranes into cytoplasm.

Basic research studies have demonstrated direct membrane translocationby way of culture mediums that mimic the acidic conditions of theendosome. In 1980, it was found that when cells in culture were exposedto diphtheria toxin in an acidic medium, the toxin would translocatedirectly into the cytoplasm. This advance was of basic scienceimportance as it simplified the study of how the toxin interacts withcell membranes,. This is a much easier task then studying the toxin'sinteraction with the membranes of an internal organelle such as theendosome. Subsequently the ability to translocate directly into thecytoplasm has been demonstrated for a number of bacterial toxins such asanthrax toxin (lethal factor and adenylate cyclase), Clostridiumbotulinum C2 toxin, Clostridium difficile toxin B, Clostridia sordelliilethal toxin and the clostridial neurotoxins.

The advantages of direct translocation on the efficiency of anamphipathic protein-conjugates has been demonstrated for the Clostridiumsordellii lethal toxin. When cultured cells were exposed to lethal toxinat pHs from 4.0 to 5.0 for only 10 minutes it increased the rate ofintoxication over 5-fold, lowered the minimal intoxicating dose by over100-fold, and allowed complete substrate modification within 2 h,instead of the 11 hours needed for the endosomal route.

Regarding clostridial neurotoxin, native and recombinant botulinumneurotoxin attaches to artificial bilipid layers coated withgangliosides, and translocate their light chains within seconds afterexposure to pH 5 on the cis side when the trans side is held at pH 7.0.In addition, acidic cell culture medium allows clostridial neurotoxin toenter cells that have no specific binding sites. For example, botulinumneurotoxin-B has been demonstrated to translocate into cultured coloncarcinoma cells and neutrophils by incubation in medium at pH 4.7.Moreover, even isolated clostridial neurotoxin light chains cantranslocate rapidly through bilipid membranes at pH 4.0. This effect isbelieved to be due to the presence of a separate amphipathic region inthe light chain.

In summary, acidic medium rapidly speeds the translocation ofamphipathic proteins-conjugates into cells they normally enter by theendosomal route, allows them to enter cells that they normally cannotenter, and in certain cases even allows the direct entry of the “cargo”molecule into cells.

Note that all the above-described experiments were performed to studyhow membrane translocation occurs or to study the intracellular effectsof specific molecules and do not teach, suggest or even anticipate theuse of acid mediated translocation in vivo.

In addition to amphipathic proteins, there are others possiblemechanisms of protein translocation into cells. Membrane transductionproteins have recently been identified that directly bind and possiblymerge with membranes and can translocate molecular cargo into cytoplasm.These proteins include part of the human immunodeficiency virus Tat,Drosophilae Antennapedia (Penetran), and Transportan (13 amino acidsfrom galanin and wasp venom mastoporan). Based on studies of theseproteins artificial membrane transduction proteins have been developedsuch as oligoarginine. Finally there are a variety of newer methodsbeing studied that involve encapsulating the bioactive cargo molecule.The use of these substances in conjunction with a toxic moiety couldsubstitute for the amphipathic moiety in all examples disclosed in thisspecification.

The present invention may be understood more fully by reference to thefollowing detailed description and illustrative examples, which areintended to exemplify non-limiting embodiments of the invention.

SUMMARY OF THE INVENTION

The invention relates to methods and compositions for improved deliveryof bioactive substances into cells within a localized part of the bodyby way of translocation agents. The invention encompasses methods ofdelivering the substance to the body part, methods to facilitate thebinding to and/or translocating of the bioactive substance across cellmembranes, and methods of protecting the bioactive substance fromneutralizing antibodies.

The invention also relates to compositions and methods of modulatingcellular function and treatment of disease in mammals comprising locallyadministering a bioactive substance—preferably, a regulated SNAREinhibitor—and a translocating agent to the mammal. Regulated SNAREinhibitors include bacterial neurotoxins, such as clostridialneurotoxins; tetanus neurotoxin; the free light chain portions ofbacterial neurotoxins and tetanus neurotoxin; and IgA protease.Translocating agents include acids, acidic environments, encapsulatingvectors, and protein transduction domains.

In one embodiment, the bioactive substance is delivered relativelynon-specifically to mammalian cells (preferably neurons) in a localizedarea of the mammal, thereby avoiding many difficulties with systemicadministration.

In another embodiment, the invention relates to facilitating the bindingof bioactive substances to mammalian cell membranes, whereupon, thebioactive substance is incorporated by way of the cell's naturalinternalization mechanisms.

In another embodiment, the invention relates to bypassing the naturalcell internalization mechanisms and translocating the bioactivesubstances directly across the cell membrane.

In still another embodiment, the invention relates to methods forcombining the bioactive substances with translocating agents or othermoieties that facilitate cell membrane binding and/or entry of bioactivesubstances into cells.

The bioactive substance for use in the invention can be any substance ormolecule that induces a biological response in mammalian cells or atherapeutic effect in a mammal. Preferably, the bioactive substance is abioactive part of a natural toxin. More preferably, the bioactivesubstance is a regulated SNARE inhibitor, most preferably, the bioactivesubstance is the light chain of a clostridial neurotoxin, preferably,the free light chain or its analogue, a protein called IgA protease.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions 1.1 Regulated SNAREInhibitors

As used herein, the term “regulated SNARE inhibitor” means any moleculethat inhibits the function of or cleaves one or more of the SNAREproteins involved in regulated exocytosis (regulated SNAREs). Regulatedexocytosis requires regulated membrane fusion, principally between aninternal vesicle (such as an endosome) and the cell membrane. Theminimal SNARE protein machinery involved in regulated exocytosis(regulated SNARE) are: (1) vSNARE, VAMP-2; and (2) the tSNAREs, SNAP-25and syntaxin-1, and the isoforms and splice variants of these proteinsas defined below.

Preferably, “regulated SNARE inhibitors” consist of light chains ofclostridial neurotoxins. These include the entire light chain or activefragment thereof from the botulinum neurotoxin serotypes A, B, C1, D, E,F and G and tetanus neurotoxin.

The amino acid sequences of the clostridial neurotoxin light chains areknown, and these sequences can be modified by addition, deletion, orsubstitution of amino acids, or otherwise chemically altered, wherebythe protein is modified but the inhibitory effect on at least oneregulated SNARE is retained. These modifications are accomplished bymethods well known in the art. “Regulated SNARE inhibitors” as usedherein, includes all such modified clostridial neurotoxin light chains.

The amino acid sequence of regulated SNARE inhibitors can be rearrangedusing recombinant techniques yielding molecules that retain regulatedSNARE inhibitory activity. The term “regulated SNARE inhibitor” as usedherein includes all such recombinantly rearranged molecules.

The term “regulated SNARE inhibitor” also includes all natural analoguesof clostridial neurotoxin light chains, such as those produced by thebacteria Neisseria gonorrhea, which produces the regulated SNAREinhibitor IgA protease. IgA protease can cleave the IgA class ofantibody as well as VAMP-2.

The term “regulated SNARE inhibitor”, as used herein, further includes:(1) proteins and other molecules that can cleave or inhibit regulatedSNAREs including non-functional fragments of regulated SNARE proteins(Apland JP et al.; Inhibition of neurotransmitter release by peptidesthat mimic the N-terminal domain of SNAP-25, J. Protein Chem. (2003)22:147-53), hereby incorporated herein by reference. (2) antibodies thatbind to regulated SNAREs Breedveld FC: Therapeutic monoclonal antibodiesLancet 2000; 355: 735-40), hereby incorporated herein by reference; and(3) antibodies and other molecules that interact with additional cellproteins in the fusion process. It is known that altered andnon-functional forms of the regulated SNAREs can interfere withregulated exocytosis. In addition, antibodies can bind to the regulatedSNAREs and inhibit their function.

Furthermore, in vivo, the regulated SNAREs interact with a many otherproteins that are involved in the fusion process, and these too can betargeted for inhibition. Proper vesicle trafficking involves vesicleformation, maturation, transport, docking, priming, fusing, andrecycling. A non-limiting list of associated proteins involved inregulated vesicle trafficking and exocytosis includes: Synaptogamin,Synaptophysins, Peptide amidase, Synapsins, Synaptogyrins, Cytochromeb561, GABA/glutamate transporters, Rab3A, B, and C, Processingpeptidases, Synaptotagmins 1 & 2 (PC1, PC2, CPE etc), SV2s,IA-2/phogrin, SVOP, SCAMPs, Synaptobrevins, Vacuolar proton pump,Cysteine string protein, Zinc transporters, Catecholamine transporters,Chloride transporter, SM proteins particularly Munc-13 and 18, and hrs.The participation of these proteins is described in various reviewarticles (Gerber S H, Sudhof T C: Molecular determinants of regulatedexocytosis, Diabetes, 2002, 51, supplement 1:s3-11, hereby incorporatedherein by reference; Mayer A: Membrane fusion in eukwyotic cells, Annu.Rev. Cell Dev. Biol. 2002. 18:289-314), hereby incorporated herein byreference.

Preferably, a regulated SNARE inhibitor is a protein, protein fragment,or conjugate thereof. Preferably, regulated SNARE inhibitors can crossfrom the extracellular fluid through the cell membrane and into the cellcytoplasm. Examples of regulated SNARE inhibitors include, but are notlimited to, amphipathic bacterial toxins, such as clostridialneurotoxins from Clostridia botulinum, berati, butyricum and tetani.More preferably, the regulated SNARE inhibitor is the light chainportion of clostridial neurotoxins. In another preferred embodiment, theregulated SNARE inhibitor is IgA protease.

1.2 Regulated SNARES

As used herein, regulated SNARE proteins are proteins involved inregulated vesicle trafficking and exocytosis. Regulated exocytosisrequires regulated membrane fusion, principally between an internalvesicle and the cell membrane. The minimal SNARE protein machineryinvolved in regulated exocytosis are: (1) vesicle SNAREs VAMP-2 ; and(2) :target SNARES: SNAP-25 and syntaxin-1. Also included under themeaning of regulated SNAREs are isoforms and splice variants of theseproteins, and putative regulated SNARES as recognized by their selectiveinhibition by one or more clostridial neurotoxins:

1.2.1 VAMP

VAMP-2, also called synaptobrevin, has two isoforms numbered 1 and 2(Rossetto O. et al. VAMP/Synaptobrevin isoforms 1 and 2 are widely anddifferentially expressed in non-neuronal tissues, J. Cell. Biol. (1996)132:167-79), hereby incorporated herein by reference. Also used hereinand included under the definition of regulated SNAREs is cellubrevin,which is an isoform of VAMP cleaved by tetanus neurotoxin (Hajduch E etal., Proteolytic cleavage of cellubrevin and vesicle-associated membraneprotein (VAMP) by tetanus toxin does not impair insulin-stimulatedglucose transport or GLUT4 translocation in rat adipocytes, Biochem. J.1997 Jan 1; 321 (Pt 1):233-8), hereby incorporated herein by reference,and botulinum toxin B (Tamori Y et al., Cleavage of vesicle-associatedmembrane protein (VAMP)-2 and cellubrevin on GLUT4-containing vesiclesinhibits the translocation of GLUT4 in 3T3-L1 adipocytes Biochem.Biophys. Res. Commun. 1996 Mar 27; 220(3): 740-5), hereby incorporatedherein by reference, and botulinum D (Cheatham B et al.,Insulin-stimulated translocation of GLUT4 glucose transporters requiresSNARE-complex proteins Proc. Natl. Acad. Sci. USA. 1996 Dec 24; 93(26):15169-73), hereby incorporated herein by reference.

1.2.2 SNAP

SNAP 25 has 2 splice variants termed SNAP 25 A and B and these areincluded under the definition, as they participate in regulatedexocytosis and are cleaved by CNT. Another example of an regulated SNAREis the putative SNAP variant G22K, that is involved in regulatedexocytosis of neutrophil white blood cell granules and is cleaved bybotulinum toxin D. (Nath J. et al., Involvement of a botulinumtoxin-sensitive 22-kDa G protein in stimulated exocytosis of humanneutrophils., J. Immunol. (1994) 152:1370-9), hereby incorporated hereinby reference.

1.2.3 Syntaxin

Syntaxin 1 has two isoforms, syntaxin 1A and 1B, and both are cleavedonly by botulinum toxin C1 (Foran P et al.: Botulinum neurotoxin C1cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffincells: correlation with its blockade of catecholamine release.Biochemistry. 1996 Feb 27; 35(8): 2630-6), hereby incorporated byreference herein.

1.3 Free Light Chains Of Bacterial Neurotoxins

As used herein, the term “free light chains” refers to the light chainsof bacterial neurotoxins or a biologically active fragment thereofwithout the heavy chain binding or translocation domains. Notably,Clostridial neurotoxins light chains contain a small translocationdomain at their N-terminal that is separate from that present on heavychains. IgA protease is also believed to have any translocation domain.Non-limiting examples of free light chains include the light chainsderived from clostridial neurotoxins, such as botulinum neurotoxinserotypes (including botulinum neurotoxin serotype is A, B, C1, D, E, Fand G), tetanus neurotoxin. Another non-limiting example is the freelight chain is IgA protease.

1.4 Translocation or Translocating

As used herein, the terms “translocation” or “translocating” withrespect to translocation of regulated SNARE inhibitors across cellmembranes means transfer of a molecule across the cell membrane. Thistransfer may or may not be accomplished by the formation of endosomes.“Direct translocation” means that the molecule passes through the cellmembrane without being internalized into endosomes.

1.5 Translocating Agent

As used herein, the phrase “translocating agent” means any substance,molecule, or environmental condition that facilitates translocation of abioactive molecule (preferably, a regulated SNARE inhibitor) across alipid membrane, preferably the outer cell membrane of a mammalian cell.Preferred translocating agents include, but are not limited to, acids oran acidic environment; amphipathic moieties (i.e., molecules orpolypeptides that become hydrophobic at pHs other than 7.4, preferablyat more acidic pH); protein transduction domains, such as cationicpolymers or polypeptides that bind to cell membranes; and encapsulationvectors, which encapsulate the bioactive substance within artificialvesicles that in turn bind or translocate the bioactive substance withinmammalian cells. Examples of encapsulating vectors include, but are notlimited to, liposomes, niosomes, transferosomes, viruses andnanoparticles.

1.6 Facilitating Translocation

As used herein, “facilitating translocation” means an increase in thenumber of bioactive moieties (preferably, regulated SNARE inhibitors,more preferably, the free light chains of bacterial neurotoxins) thatcross a cellular membrane in a specified time period with the assistanceof a translocating agent relative to the number of bioactive moietiesthat cross the cellular membrane for that time period in the absence ofthe translocating agent. “Facilitating translocation” also means adecrease in time required for a specified number of bioactive moietiesto cross the membrane with the assistance of a translocating agentrelative to the time that the specified number of bioactive moietiescross the cellular membrane in the absence of the translocating agent.

Whether translocation has been facilitated can be determined by assayswell known in the art, for example, by measuring one or more of: cellsurvival (Williamson L and Neale E: Syntaxin and 25-kDasynaptosomal-associated protein: Differential effects of botulinumneurotoxin C1 and A on neuronal survival; J Neurosci Res 52:569-583(1998), hereby incorporated herein by reference; cell division (Conner S& Wessel GM: Syntaxin Is Required for Cell Division Molecular Biology ofthe Cell, Vol. 10, 2735-2743, August 1999), hereby incorporated hereinby reference; by membrane replacement (Cheatham B et al.,Insulin-stimulated translocation of GLUT4 glucose transporters requiresSNARE-complex proteins., Proc. Natl. Acad. Sci USA, 1996, 93:15169-73),hereby incorporated herein by reference; or by exocytosis (Keller J. etal., Uptake of Botulinum Neurotoxin into Cultured Neurons, Biochemistry2004, 43, 526-532), hereby incorporated herein by reference.

1.7 Cell Membrane Binding

As used herein, the term “binding” with respect to binding of moleculesto cell membranes means that the molecule bonds non-covalently to themembrane or the exposed proteins, lipid or carbohydrate moieties ofmembrane embedded molecules.

1.8 Cell Membrane Fusion

As used herein, “fusion” means that the merging of lipid membranes orthat hydrophilic moieties of a molecule have become embedded into themembrane.

1.9 Amphipathic Moieties or Proteins

As used herein, the terms “amphipathic moieties” or “amphipathicproteins” means natural (wild type) or artificially produced moieties orproteins that are capable of changing from hydrophilic to hydrophobicsecondary to changes in pH, either increased acidity or increasedalkalinity. Preferably in the hydrophobic state at least part of themolecule can fuse with lipid membranes.

1.10 Amphipathic Protein Conjugates

As used herein, the term “amphipathic protein conjugate” means amolecule or molecule complex comprising an amphipathic moiety oramphipathic protein (as defined above) that is associated with abioactive substance and that is capable of translocating the bioactivesubstance into the cytoplasm of a cell, preferably, at acidic pH.

1.11 Local Delivery or Local Administration

As used herein, the terms “local delivery” or “local administration”,with respect to delivery of a regulate SNARE inhibitor to a mammal,means delivery or administration of the regulated SNARE inhibitor to alocal part of the body, through or into the skin or mucosa of a mammal.Preferably, local delivery is effected without significant absorption ofthe regulated SNARE inhibitor into the mammal's blood stream withsubsequent systemic distribution. The purpose of local delivery is toelicit a local affect in the area (selected site) of administration.Preferably, local delivery or administration is by way of apharmaceutically effective formulation.

1.12 Therapeutically Effective Amount

The term “therapeutically effective amount” with respect to a regulatedSNARE inhibitor means an amount of the regulated SNARE inhibitor,preferably, a non-toxic amount, sufficient to treat, prevent, or reducethe occurrence or magnitude of symptoms of a disease or medicalcondition being targeted. Preferably, when administered to a mammal, atherapeutically effective amount is administered in a pharmaceuticallyeffective formulation.

1.13 Modulating Cellular Function

As used herein the phrase “modulating cellular function” means anychange in a cell's function, preferably, a change due to a change in therate of the number of membrane fusions occurring in the cell, morepreferably, a decrease in said fusion events, and most preferably adecrease in regulated membrane fusion occurring as part of regulatedexocytosis.

1.14 Modulatorily Effective Amount

The term “modulatorily effective amount” with respect to a regulatedSNARE inhibitor means an amount of the regulated SNARE inhibitor,preferably a non-toxic amount sufficient to modulate cellular functionwhen locally administered to a mammal. Preferably, when administered toa mammal, a modulatorily effective amount is administered in apharmaceutically effective formulation.

1.15 Wild Type

As used herein, the term “wild type” or “natural toxins” means naturallyfound proteins and toxins from plants, animals, or microbes. Numerousmodifications and alterations can be made to the wild type molecule. Atpresent, recombinant techniques allow for the formation of a clostridialneurotoxin molecule in which any part of the molecule can be replaced,either by an analogous part of another clostridial neurotoxin, or by acompletely different molecule. In addition the protein can be chemicallymodified in various ways to decrease immunogenicity, decrease diffusionfrom the site of application, increase binding, biological persistence,toxicity or for other reasons. For example, the light chain of botulinumneurotoxin/A has amino acid fragments for various secondary modificationsites (hereinafter “modification sites”) including, but not limited to,N-glycosylation, casein kinase II (CK-2) phosphorylation, N-terminalmyristylation, protein kinase C (PKC) phosphorylation and tyrosinephosphorylation (U.S. 2002/0127247A1: Modified clostridial neurotoxinswith altered biological persistence), hereby incorporated herein byreference. Nucleic acid itself can be used in place of protein andinserted into cells for later translation into protein. (WO 14570Recombinant Activatable Neurotoxins), hereby incorporated herein byreference. Regulated SNARE inhibitors include all these changes andmodifications.

1.16 Chimeric Or Hybrid Toxin

As used herein, the term “chimeric” or “hybrid toxin” refers tobioactive molecules either created by joining parts derived from two ormore natural toxins (U.S. Pat. No. 6,444,209: Hybrid botulinalneurotoxins, hereby incorporated herein by reference) or created byjoining all or part of a natural toxin with all or part of another largemolecule, such as an antibody. In the wild, it has been found that somestrains of Clostridia produce a hybrid toxin composed of proteins chainsfrom two different serotypes. But with recombinant genetics this can beaccomplished artificially. Therefore, the light chain of any CNT can becombined with the heavy chain of any other. For example, a chimerictoxin consisting of the “heavy” (ca. 100,000 MW) chain of botulinumtoxin and the “light” (ca. 50,000 MW) chain of tetanus toxin wasconstructed and found to have six times the potency of native tetanustoxin (Weller, U. et al., “Cooperative Action of the Light Chain ofTetanus Toxin and the Heavy Chain of Botulinum Toxin Type A on theTransmitter Release of Mammalian Motor Endplates” Neurosci. Letters(1991) 122: 132-134), hereby incorporated herein by reference.

1.17 Local Tissue Acidification

As used herein, the term “Acidification” means creation of acidicconditions in mammalian tissue in relation to the neutral pH 7.4 ofextracellular fluid. “Acid” or “acidic solution” means apharmaceutically safe solution or other carrier that can be used todecrease the pH, preferably to facilitate translocation of amphipathicmoieties or proteins. “Buffered acid solutions” as used herein mean theinclusion of a buffer that serves to maintain the solution at a desiredpH, preferably, the pH that is most preferable for translocation of theregulated SNARE inhibitor.

1.18 “Units of Toxin”

As used herein, the phrase “units of toxin” means the amount that causesdeath in 50% of 20 gram Swiss Webster mice upon injection. As usedherein when “units” refer to an entity that cannot cause animal death bythemselves, such as compositions of clostridial neurotoxin light chains,it refers to the molar equivalent of light chains that is obtained fromthe dose of whole toxin that can be assayed. As an illustrative exampleif a unit of a particular botulinum toxin serotype weighs 6 nanograms,than a unit of botulinum toxin of light chain composition weighs 2nanograms. A “unit” of IgA protease is fifty times more by weight than aunit of tetanus neurotoxin light chains as defined by a biological assayof regulated SNARE inhibition of nor-epinephrine by chromaffin cells(Binscheck T et al., IgA protease from Neisseria gonorrhoeae inhibitsexocytosis in bovine chromaffin cells like tetanus toxin, J. Biol. Chem.1995 Jan 27;270(4):1770-4), hereby incorporated herein by reference.

2. Sources of Regulated Snare Inhibitors

Regulated SNARE inhibitors are readily commercially available, forexample, botulinum toxin serotypes A, B, C1, D, E, F, G, tetanus toxinand their light chains are available from List Biological Laboratories(www.listlabs.com) and/or Wako Labs (Japan) and Metabiologics, Inc.,Madison Wis.

Botox™ (botulinum type A) is available from Allergan Corporation, IrvineCalif.; Myobloc™ (botulinum toxin type B) is available from Elan,Dublin, Ireland; Dysport™ (botulinum toxin A) is available from IpsenSpeywood, Bath, United Kingdom; IgA protease is available from CliniquaCorporation, Fallbrook, Calif.

3. Methods of the Invention for Translocating and/or Binding RegulatedSnare Inhibitors

In one embodiment, the invention provides methods for binding regulatedSNARE inhibitors to the cell membrane and/or translocating regulatedSNARE inhibitors across the cell membrane. In one embodiment, themethods of the invention are directed to acid mediated translocation. Inanother embodiment, the methods of the invention are directed totranslocation and binding of regulated SNARE inhibitors by way ofprotein transduction domains. In yet another embodiment, the methods ofthe invention are directed to translocation and binding of regulatedSNARE inhibitors by way of encapsulation vectors.

In still another embodiment, the methods of the invention are directedto specific binding to or translocating, i.e., the methods of theinvention are such that the regulated SNARE inhibitors target specificsub-populations of cells in a local tissue. In another embodiment, themethods of the invention are directed to non-specific binding ortranslocating, i.e., the methods are such that the regulated SNAREinhibitors bind to or are translocated into all cells in a local tissue.

In another embodiment, the methods of the invention are directed totranslocating or binding the light chains of regulated SNARE inhibitorsinto cells non-specifically. This is therapeutically beneficial as thelight chains are specific inhibitors of regulated exocytosis. As thelight chains cleave one of the three regulated SNARE proteins thisdifference among them can be used to therapeutic advantage. Morespecificity can be achieved by controlling the volume of tissue to whichthe light chains are delivered.

In one embodiment of the invention, regulated SNARE inhibitors areinjected from about one-second to about one hour prior to acidification,thereby allowing regulated SNARE inhibitors to bind prior totranslocation. In another embodiment, injection of regulated SNAREinhibitors is performed in conjunction with injection of an acidformulation, thereby causing more general and non-specific binding. Theinjection of acid solution could also precede injection of the proteinof the inventions.

3.1 Acid Mediated Translocation Of Regulated SNARE Inhibitors

In one embodiment, the invention provides methods for facilitating thebinding and/or direct translocation of regulated SNARE inhibitors uponlocal delivery of regulated SNARE inhibitors to mammalian tissue havingan acidic extracellular environment. Depending on the identity of or thedelivery composition of the regulated SNARE inhibitor, its binding andtranslocation can be facilitated at different pH values.

3.1.1 Generation of an Acidic Extracellular Environment

-   -   3.1.1.1 Use Of An Acidic Carrier Solution To Generate An Acidic        Extracellular Environment

In one embodiment of the invention, regulated SNARE inhibitors areadministered with an acidic medium, either simultaneously (for example,mixed in an acidic formulation) or consecutively, to effecttranslocation of regulated SNARE inhibitors into the cell cytoplasm inthe local area of injection. This works efficiently with neuronal cellsas they contain binding sites but is also effective with non-neuronalcells.

For use in the methods of the invention, preferred pH values of the siteof local administration range from about 3.5 to about 7, preferably fromabout 3.5 to about 4.5 for isolated or free neurotoxin light chains,and, preferably, from about 4.5 to about 6, more preferably, about 5 toabout 5.5 for wild type whole CNT.

Acidification in local tissue regions can be the result of localcellular metabolism, the application of acids or acidic solutions, orthe formation of hydrogen ions by electrical energy. Examples of acidicsolutions suitable for use in the invention include, but are not limitedto, phosphoric, acetic, hydrochloric, lactic or hyaluronic acids. Thesecan be in aqueous solution, gels, biodegradable and non-degradablematerials. Further examples of pharmacologically suitable acids andbuffers are well known in the art and can be found in Goodman andGillman's The Pharmacologic Basis for Therapeutics, 10^(th) edition,McGraw Hill, 2001, hereby incorporated herein by reference.

-   -   3.1.1.2 Buffered Compositions of Acidic Solutions

Physiological solutions contain significant amounts of buffer,substances that oppose shifts in pH. Buffers work by absorbing ordonating protons to oppose shifts in an acidic or alkaline direction,respectively. The ability of a buffer to oppose a shift in pH depends onits pK and its concentration. The pK corresponds to pH at which equalamounts of the protonated and non-protonated versions of the buffer arepresent. The concentration of a buffer is preferably described as theamount of protons it can buffer, expressed as milli-equivalents perliter of solution. At the pK the buffer has equal capacity to opposeeither an acidic or alkaline shift in pH. However, at the pK only halfof the total capacity of a buffer is available to oppose a pH shift. Ifa buffer has a low pK (acid) and the pH of the solution is high(alkaline) most of the buffer is unprotonated and it has little capacityto resist further shifts toward alkalinity. However, in this examplemost of the capacity of the buffer is available to oppose pH shifts inthe acidic direction, which is usually preferable in body tissues.

In one embodiment of the invention, the acid solution is buffered with apK at the most appropriate pH that facilitates the translocation of theregulated SNARE inhibitor. primary buffer in extracellular fluids is

-   -   3.1.1.3 Electrical Stimulation to Generate an Acidic        Extracellular Environment

It is one embodiment of this invention that electricity be used to aidin transferring AP-cargo regulated SNARE inhibitor across membranes,preferably to directly translocate the regulated SNARE inhibitor fromthe extracellular fluid into a cell, into lipids, and across lipidmembranes.

Electrical stimulation current within body fluid causes a chargegradient between the anode and cathode electrodes that attracts chargedmolecules. At relatively high currents electrochemical reactions occurat both the anodal and cathodal electrode surfaces. At the anodalelectrode H+ ions are formed, lowering the pH. At the cathodal electrodeOH− ions are simultaneously formed raising the pH. Electricity may bedirect current or more preferably it is separated into individualpulses. Although the pulse voltage and/or current may vary preferablythe pulse has a square shape. Specifically, at the initiation of thepulse voltage rises instantaneously to its peak level, stays at the peaklevel for the pulse duration and then instantaneously drops to zero atthe end of the pulse.

The extent of the pH change, and the volume in which it occurs, isrelated to the variables of the electrical voltage and amperage. Thesevariables are preferably expressed as the charge density, and mostpreferably as amperage per square millimeter of electrode surface.

Variables of electrode shape, composition, surface texture andorientation all effect the charge density and generation of protons.Electrodes can be made of any conduction material, metal, gel orelectrolytic solution and a vast range of possibilities are known in theart (Bockris J., Modern Electrochemistry 1 Ionics, Plenum Press NewYork, 1970), hereby incorporated herein by reference. Preferable metalsare iridium, platinum, gold, stainless steel or tungsten, and mostpreferred is oxidized iridium as it forms hydrogen ions at a wide rangeof charge densities (Ballestrasse C. L. et al., Calculations of the pHchanges produced in body tissue by a spherical stimulation electrode,Annals of Biomedical Engineering, (1985) 13:405-424), herebyincorporated herein by reference.

A non-limiting example of an anodal electrode is a spherical ball ofoxidized iridium with a radius of 1 μm. Electrical stimulation isdelivered by square wave pulse lasting 0.2 seconds and the currentdensity is 4 mAmps/mm2. Under these conditions the neutral pH at thesurface of electrode falls to pH 4. This is sufficient for the inventionto directly translocate a clostridial neurotoxin light chain across cellmembranes. The pH change is directly proportional to distance, with pH 5at 1 μm from the electrode surface and no significant change in pH 4 μmaway from the surface (Ballestrasse C. L. et al., Calculations of the pHchanges produced in body tissue by a spherical stimulation electrode,Annals of Biomedical Engineering, (1985) 13:405-424), herebyincorporated herein by reference.

This non-limiting example is illustrative of an embodiment of electricalinduced acidification when precise localization of acidity is preferred.Essentially, the acidity is produced adjacent to the electrode,therefore the clinician can control the tissue volume in which theacidity is produced.

In one non-limiting example, the metallic shaft of a hollow bore needleinsulated on the exterior surface, except at the opening at the tip, isconnected to the anode of an electricity source, while a distant cathodeelectrode is attached to the skin surface. The needle is attached to asyringe containing a protein of the invention and an acidic electrolyticsolution. A current is passed (continuous or pulsed) through the needleelectrode to form protons in the immediate area of the needle tip. As aresult, the local area around the needle tip has the proper acidity tocause translocation. Even if the tissue has been injected with a proteinthat normally would not enter cells in the area upon injection, theelectricity causes local acidity that allows translocation. As furtherillustration of the above embodiment of the invention, the sameelectrode surface described above is exposed at the very tip of a thinneedle attached to a syringe containing a clostridial neurotoxin lightchain in a physiological saline solution. A patient has severe recurrentepileptic seizures originating from a 1 mm² lesion on the surface of thecerebral cortex. During a neurosurgical procedure the lesion is exposedand the needle is placed into the lesion where 0.01 cc of clostridialneurotoxin light chain solution is injected simultaneously with one ormore 0.2 msec anodal electrical pulses. The pulses generate protons andresultant requisite acidity in the lesion without disturbing braintissue less than 1 mm away. Although clostridial neurotoxin light chainsdo not normally enter cells, including neurons, the invention causesthem to translocate directly in the pathological neurons of the lesion.The ability of electricity to increase the local acidity in preciselylocalized target tissue is of critical importance in the CNS as so manyvital structures are often adjacent to pathological lesions,.

3.1.2 Direct Translocation of Wild Type Clostridial Neurotoxin

Wild type botulinum toxins naturally bind to cholinergic efferentneurons and epithelial cells where they are internalized by endocytosis.In contrast there are some low-affinity gangliosides on all neuronalmembranes and, although botulinum toxin binds to these sites, it is notinternalized under normal conditions (M. V. De Angelis et al.: Anti-GD1aantibodies from an acute motor axonal neuropathy patient selectivelybind to motor nerve fiber nodes of Ranvier, 121 JOURNAL OFNEUROIMMUNOLOGY 79-82 (2001), hereby incorporated herein by reference.However, according to one embodiment of the invention exposure of themembrane bound botulinum toxin to an acidic extracellular environment,for example, a pH range from about 4.5 to 6, more preferably, a pH valueof about 5 to 5.5 triggers direct translocation of the free light chainacross the cell membrane. Thus, according to this embodiment of theinvention, light chains are delivered into neurons that botulinumneurotoxin cannot normally enter, including sympathetic neurons andsensory neurons. Particularly notable are the subclass of sensoryneurons that sense pain. As discussed in more detail below, this greatlyincreases the efficacy of regulated SNARE inhibitors to treat disease.

In contrast to botulinum toxin, tetanus toxin binds to and isinternalized by all peripheral neurons. However, the internalizedtetanus toxin is preferentially transported to the CNS where itsselective block of inhibitory neurons causes excitation. Thus, accordingto another embodiment of the invention, where a decrease of peripheralneuron activity is beneficial, the tetanus toxin can be exposed to anacidic environment before it is internalized. Into endosomes. Exposingthe bound tetanus toxin to an acidic extracellular environment,preferably from about 4.5 to 6, and more preferably from about pH 5 to5.5 triggers direct translocation of the tetanus toxin light chainacross the cell membrane. This demonstrates how the method of thisinvention changes the increased activity that normally occurs in neuronsintoxicated with tetanus toxin into decreased activity.

Under ideal conditions clostridial neurotoxins bind and are internalizedwithin seconds. However, in vivo conditions are rarely ideal. Afterinjection, the neurotoxins must diffuse through tissue to reach theirnatural high affinity binding sites on the presynaptic membrane. Becausethe toxins are large molecules, this diffusion can be quite slow.Therefore, depending on the particular tissue, the time allowed frominjection of toxin to acidification can vary from simultaneousadministration to up to ten hours, although 2 hours is preferred, and 20minutes is more preferable. The exact time varies with the specifictissue and mode of delivery. However, after endocytosis begins, thetoxin is no longer exposed to the extracellular environment and,therefore, is less effected by extracellular acidification.

4. Encapsulation Vectors

In another embodiment of the invention, regulated SNARE inhibitors aretranslocated across the cell membrane by an encapsulation vectors. Asused herein the terms “encapsulation vectors” or “vectors” meanpharmaceutical preparations that physically or chemically surround theregulated SNARE inhibitor and function to facilitate translocation.Preferably the regulated SNARE inhibitor is not covalently bonded to thevector. Both physical and chemical encapsulation vectors are suitablefor use in the invention.

Examples of physical encapsulation vectors suitable for use in theinvention include, without limitation, liposomes, niosomes, viruses andnanoparticles.

In one aspect of this embodiment, the encapsulating vesicle is aliposome. Liposomes are artificial vesicles with single or multiplemembranes that encapsulate surround a bioactive cargo and deliver it tocells either by membrane fusion or endocytosis. The membranes are madefrom natural and synthetic phospholipids, glycolipids, and other lipidsand may include cholesterol; charged species which impart a net chargeto the membrane; and specific binding moieties on their surface; andother lipid soluble compounds which have chemical or biologicalactivity.

Preferred liposomes for use in the invention can be triggered to releasetheir contents or fuse in response to pH stimuli, as they canpotentially respond to acidic environments in vivo. Such environmentsinclude those encountered in tumor tissue and primary endocyticvesicles. See e.g., Hafez Tunable pH-Sensitive Liposomes Composed ofMixtures of Cationic and Anionic Lipids 79 BIOPHYSICAL JOURNAL 1438-1446(2000), hereby incorporated herein by reference.

Botulinum toxin free light chain can be delivered via liposomes to blockin vitro neuromuscular transmission at nanomolar doses, Paiva A, Dolly JO. Light Chain Of Botulinum Neurotoxin Is Active In Mammalian MotorNerve Terminals When Delivered Via Liposomes FEBS Lett. 1990 Dec 17;277(1-2): 171-4, hereby incorporated herein by reference; WO03/101483A1, Pharmaceutical Preparation Of Botulinum Neurotoxin, MethodsOf Synthesis And Methods Of Clinical Use, hereby incorporated herein byreference, discloses methods of producing liposomes containing botulinumtoxin.

In one preferred embodiment of the invention, liposomes are used as atranslocating agent to skin cells and certain liposomes are appropriatefor transdermal drug delivery (see e.g., U.S. Pat. No. 5,190,762 MethodOf Administering Proteins To Living Skin Cells, hereby incorporatedherein by reference).

In another preferred embodiment, liposomes can deliver regulated SNAREinhibitors to cancer cells (Harrington K et al., Liposomally TargetedCytotoxic Drugs For The Treatment Of Cancer, Journal of Pharmacy andPharmacology 2002, 54: 1573-1600, hereby incorporated herein byreference).

In another aspect of this embodiment, the encapsulating vesicles areniosomes. Niosomes, which may be considered a special case of liposomes,are prepared from non-ionic surfactants such as polyoxyethylenealkylether, polyoxyethylene alkylester or saccharose diester, see e.g.,Rentel C O et al: Niosomes As A Novel Peroral Vaccine Delivery SystemInt. J. Pharm. 1999 Sep 20;186(2):161-7), hereby incorporated herein byreference.

In another aspect of this embodiment, the encapsulating vesicles areviruses, protein or glycoprotein structures encapsulating regulatedSNARE inhibitor. See e.g., Cullen, B. R. Journey to the center of thecell. Cell 105, 697 (2001), hereby incorporated herein by reference.

In another aspect of this embodiment, the encapsulating vesicles arenanoparticles. Nanoparticles are 10 to 1000 nm particles made frompolymers with the bioactive agent embedded inside. A preferredembodiment are nanoparticles composed by polyDL-lactide-coglycolide.These particles enter into cells by non-specific pinocytosis. Theyescape from acidified endosomes into cytoplasm and slowly dissolve andrelease their bioactive cargo. The polymeric material dissolves intolactic and glycolic acids that are metabolized by the cell andeliminated. See e.g., Panyam J et al., Rapid Endo-Lysosomal Escape OfPolydl-Lactide-Coglycolide Nanoparticles: Implications For Drug And GeneDelivery FASEB J. (2002) 16, 1217-1226, hereby incorporated herein byreference; Labhasetwar, V. (1997) Nanoparticles For Drug Delivery,Pharm. News 4, 28-31, hereby incorporated herein by reference.

Chemical encapsulation vectors are molecules that bond to the regulatedSNARE inhibitor either by covalent or non-covalent bonds and isolate theregulated SNARE inhibitor from the surrounding aqueous environment. Byanalogy an illustrative example of the concept is the wild typebotulinum toxin as produced by Clostridia in contaminated food.Botulinum toxin A is actually produced by bacteria with a coating ofprotective proteins that surround the toxin. These proteins are thoughtto protect the toxin from the acidic environment of the stomach whichcan reach a pH of 2.0 and denature the toxin. Another example of achemical vector are monoclonal antibodies that are produced to bind tovarious antigenic sites of a toxin. The monoclonal antibodies aredesigned to protect the toxin from the extracellular environment butdissociate from the toxin at the acidic pH such as found in endosomesand the methods of this invention, see e.g., Raso V et al.,Intracellular Targeting with Low pH-triggered Bispecific AntibodiesJournal of Biological Chemistry, 1997, 272:27623-27628, herebyincorporated herein by reference. Another chemical encapsulating vectoris the protein transduction domain Pep-1, or its equivalent, that isdisclosed below.

5. Translocation and Binding of Regulated Snare Inhibitors by Way ofProtein Transduction Domains

In another embodiment, the invention relates to translocation ofregulated SNARE inhibitors across cell membranes by way of proteintransduction domains. Protein transduction domains are relatively shortpolypeptides that, generally, bind to cells non-specifically (i.e., donot differentiate between cell types). It is believed that the mechanismof action involves electrostatic attraction between cationic chargedamino acids of the protein transduction domain and anionic charges onthe cell surface. These cause non-specific binding and internalization.Although the mechanism by which the protein domain enters cells on itsown is unclear, when conjugated to bioactive cargo it enters viaendosomes. Acidification causes direct translocation across themembrane. See e.g., Leifert J and Whitton L: “Translocatory Proteins”and “Protein Transduction Domains”: A Critical Analysis of TheirBiological Effects and the Underlying Mechanisms, Molecular therapy,2003, 8:13-20, hereby incorporated herein by reference; Lindsay M.,Peptide-Mediated Cell Delivery: Application In Protein Target ValidationCurrent Opinion in Pharmacology 2002, 2:587-594, hereby incorporatedherein by reference; U.S. 2002/0098236A1, Transport Vectors, herebyincorporated herein by reference.

Studies of the minimum translocation region identified a positivelycharged section between amino acids 47 and 57, which was previouslyassociated with DNA binding, Vives E, Brodin P. Lebleu B, A TruncatedHIV-1 Tat Protein Basic Domain Rapidly Translocates Through The PlasmaMembrane And Accumulates In The Cell Nucleus. J Biol Chem 1997,272:16010-16017, hereby incorporated herein by reference. Similarstudies of antennapedia, a Drosophila homeodomain transcription factor,identified a 16-amino-acid PTD derived from region 43-58, and alsolocated within the DNA-binding third domain. Lindgren M et al.,Translocation Properties Of Novel Cell Penetrating Transportan AndPenetratin Analogues. Bioconjug. Chem. 2000, 11:619-626, herebyincorporated herein by reference. Since these initial observations, ahost of short peptides have been identified and shown to rapidlytranslocate across membranes. However, conjugation and delivery ofbiological cargo has been predominantly performed using the peptidesderived from TAT, antennapedia and transportan, a synthetic chimeraderived from galanin and mastoparan (Pooga M, Hallbrink M, Zorko M,Langel U: Cell Penetration By Transportan FASEB J1998, 12:67-77, herebyincorporated herein by reference. Comparison of the uptake rate byfluorescence resonance energy transfer (FRET) analysis showed that theuptake of all these protein transduction domains was essentiallycomplete within 15-60 min.

A preferable protein transduction domain is Pep-1 marketed as Chariot™(Active Motif, Carlsbad Calif.) Pep-1 binds to bioactive cargo vianon-covalent interactions, functions like a chemical encapsulation toprotect the cargo extracellularly and then can translocate bioactivecargo intracellular in vitro and in vivo with 65-90% efficiency Pep-1 iscomposed of 21 amino acids consisting of three domains: (1) ahydrophobic tryptophan-rich motif containing five tryptophan residuesrequired for efficient targeting to the cell membrane and for forminghydrophobic interactions with proteins; (2) a hydrophilic lysine-richdomain (KKKRKV) derived from the nuclear localization sequence (NLS) ofsimian virus 40 (SV-40) large T antigen, required to improveintracellular delivery and solubility of the peptide vector; and (3) aspacer domain (SQP), separating the two domains mentioned above,containing a proline residue, which improves the flexibility and theintegrity of both the hydrophobic and the hydrophilic domains (Morris Met al: Peptide carrier for the delivery of biologically active proteinsinto mammalian cells, 2001 19:1173-1176) Chariot™ User Manual (versionA), hereby incorporated by reference herein, teaches how to combinePep-1 with proteins prior to application to cells and is hereinincorporated by reference in its entirety. In vivo use of Pep-1 inmammals is disclosed in, Aoshiba K, Alveolar Wall Apoptosis Causes LungDestruction and Emphysematous Changes American Journal of RespiratoryCell and Molecular Biology. Vol. 28, pp. 555-562, 2003, herebyincorporated herein by reference.

According to this embodiment of the invention, protein transductiondomains are particularly useful with certain regulated SNARE inhibitors,such as the free light chain (which do not contain the heavy chainbinding region) of clostridial neurotoxins and the protein IgA protease,that normally do not bind efficiently, if at all, to cell membranes. Theprotein transduction domain assists in bringing such molecules acrossthe cell membrane where they can perform their SNARE inhibitingfunction.

6. Methods for Drug Delivery to Minimize Production of Antibodies

Immunogenicity is a major problem for complex pharmaceuticals. If apharmaceutical is antigenic (generates antibodies), the drug should beused as efficiently as possible, to minimize neutralizing antibodies.

Antibodies circulate through the circulation and pass into theextracellular fluids. In most tissues, of antibody levels in theextracellular fluids is approximately the same as in plasma, except forthe cerebrospinal fluid where antibodies are removed by an activeprocess. If antibodies against a particular drug exist, it is importantto minimize the drug's exposure.

According to one embodiment of the invention, if antibodies exist or aregenerated in increased amounts, certain modifications are made in themolecule itself or in the method of delivery to minimize antibodyproduction and, to the extent antibodies are present, to minimizeexposure of the drug to these antibodies.

For example, the production of neutralizing antibodies can be mitigatedby decreasing the amount of drug used for a given condition andadminister the drug such that it binds to its target as quickly aspossible, thereby minimizing the extra drug that diffuses away from thearea.

In another embodiment of the invention, the acid mediated translocationdescribed above helps accomplish all these goals. Acidic environmentsinterfere with antibody function. Tumors and inflamed tissue are bothtissues where antibody function is known to be impaired. Acid decreasesthe affinity of antibodies to antigens, perhaps by making conformationalchanges in both. Therefore, acid mediated translocation directly aidsthe injected drug in avoiding neutralizing antibodies. Similarconformational changes can be achieved with extracellular alterations inionic strength, urea concentration, and other chemical manipulationswell known to those skilled in the art.

In one embodiment, regulated SNARE inhibitors, particularly, clostridialneurotoxin, can be chemically altered to cover its antigenic sites, forexample, by polyethylene glycolation (PEG). Polyethylene glycol ispositioned at various points of the molecule that prevent antibodiesfrom approaching close enough to bind without stopping the activity ofthe molecule. In experimental animals, modification of a Pseudomonasexotoxin-derived immunotoxin with monomethoxy-polyethylene glycol (mPEG)diminished immunogenicity 5- to 10-fold, prolonged circulation time andpreserved its anti-tumor effect.

In another embodiment, the amino acids side chains of regulated SNAREinhibitors are altered. This is readily accomplished by one of skill inthe art by adapting the methods disclosed in U.S. pat. Appl. Pub.2002/0127247A1 (published Sep. 12, 2002), which is hereby incorporatedherein by reference.

Another method of the invention for covering/blocking the antigenicsites is with antibodies that are released under acidic conditions, suchas are found in the endosomes.

In another embodiment of the invention, regulated SNARE inhibitors, forexample, clostridial neurotoxin, are enveloped in an immunoliposome thatis internalized into the cell by endocytosis, and then merges with themembrane of the endosomes at acidic pH, thereby liberating its contentsinto the cytoplasm.

In still another embodiment of the invention, antibodies can be useful.That is, antibodies against the drug can be injected within a period oftime before or after drug delivery thereby “soaking up” excess drug thatmight cause the induction of an immune response.

In yet another embodiment of the invention for mitigating antibodyformation, the extracellular space and/or the circulation in the area isdecreased to prevent delivery of antibodies. For example, limb can beelevated and a tourniquet applied. Cold and pressure both decreasecirculation as well as extracellular volume in tissue. Vasoconstrictorscan decrease circulation to very low levels, and tissues can toleratethis for an hour or more. Epinephrine and similar vasoconstrictors areuseful for this purpose.

In another embodiment, other substances or molecules can be injectedalong with the drug to bind antibodies. Certain bacterial proteins bindantibodies, these include proteins A, G, L, M. In addition anti-idiotypeantibodies can be injected with the drug, or at a minimum the Fabfragments. An excess of haptens can be injected to saturate antibodybinding sites. Alternatively inactive drug or fragments thereof can beused. Of this embodiment of the invention an example is to injecttetanus toxoid along with tetanus toxin.

Staphylococcus aureus protein A binds to IgG via surfaces in theFc-fragment of the heavy chain of the IgG-molecule, however, protein Alacks the ability to bind to human IgG3. Protein G binds to heavy chainsin human IgG and to all four of its subclasses. Protein H binds to theFe-fragment in IgG from human beings, monkeys and rabbits. Protein Gbinds to heavy chains in human IgG and to all four of its subclasses.Protein M binds to the Fc-fragment in IgG from humans, monkeys, rabbits,goats, mice and pigs (PCT/SE91100447), hereby incorporated herein byreference. Protein L binds to the light chains in immunoglobulins fromall of the classes of G, A, M, D and E is known. See e.g., U.S. Pat. No.4,876,194 (issued Oct. 24, 1999), which is hereby incorporated herein byreference. U.S. patent application publication no. 2003/0027283A1(published Feb. 6, 2003), which is hereby incorporated herein byreference, discloses a recombinant protein L that can be used for abovepurposes. In addition, in some autoimmune diseases, there is a greatneed to reduce or eliminate serum antibodies, and this applicationsuggests that this recombinant protein L may be of value. . Theabove-mentioned proteins are used in the analysis, purification andpreparation of antibodies from solution ex vivo for diagnostic andbiological research and have not been used in vivo.

Alternatively monoclonal antibodies can be produced that bind humanantibodies. EP0163141B1, Monoclonal anti-human IgG antibody and processfor preparing the same, hereby incorporated herein by reference, teacheshow these can be prepared, although the purpose in the disclosedinvention is for these antibodies to be part of an in vitro diagnostictest. If antibodies are to be used in humans they must be “humanized”,i.e., have any foreign antigens removed. Recombinant techniques allowfor the design and mass production of various size humanized monoclonalantibodies against selected antigens. For the purposes of the presentinvention the entire antibody against human antibody need not be used.Instead, only the binding fragment with specificity against the bindingsite of human antibodies is necessary.

7. Methods of Administration of Toxins Formulations of the Invention

Preferably, the regulated SNARE inhibitors used according to the methodsof the invention are administered by local delivery or administration bymethods well known in the art. Suitable local administration methodsinclude intraperitoneal, injection into an organ, intramuscular,intraventricular, subcutaneous, topical, sublingual, nasal, parenteral,ocular, intradermal, subcutaneous, and topical administration modes.

Preferably, the regulated SNARE inhibitors are administered inpharmaceutically acceptable formulations. Preferably, such formulationsare sterile. Pharmaceutically acceptable methods and formulations arewell known in the art, for example: parenteral injection andformulations are discussed in 2 REMINGTON: THE SCIENCE AND PRACTICE OFPHARMACY 1524-1548 (Alfonso R. Gennaro ed., 19th ed., 1995), herebyincorporated herein by reference; topical formulations andadministration is described in 2 REMINGTON: THE SCIENCE AND PRACTICE OFPHARMACY 866-885 (Alfonso R. Gennaro ed., 19th ed., 1995), herebyincorporated herein by reference; administration by way of solutions,emulsions and extracts is discussed in 2 REMINGTON: THE SCIENCE ANDPRACTICE OF PHARMACY 1495-1523 (Alfonso R. Gennaro ed., 19th ed., 1995),hereby incorporated herein by reference; administration by way ofophthalmic formulations is discussed in 2 REMINGTON: THE SCIENCE ANDPRACTICE OF PHARMACY 1495-1523 (Alfonso R. Gennaro ed., 19th ed., 1995),hereby incorporated herein by reference; administration by way ofaerosols is discussed in 2 REMINGTON: THE SCIENCE AND PRACTICE OFPHARMACY 1676-1692 (Alfonso R. Gennaro ed., 19th ed., 1995), herebyincorporated herein by reference; administration by way of powders isdiscussed in 2 REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 1598-1614(Alfonso R. Gennaro ed., 19th ed., 1995), hereby incorporated herein byreference.

Regulated SNARE inhibitors can be administered by standard techniqueswell known in the art, for example, without limitation, in formulations,such as solutions, powders, bi gels, polymers, microparticles, liposomesor micelles.

7.1.1 Administration by Local Injection

-   -   7.1.1.1 Application of Clostridial Neurotoxins along Axons

Notably, both botulinum and tetanus toxin normally bind and areinternalized into neurons at the synapse, the point of contact betweenthe neuron and its target cell. This area contains the regulated SNAREprotein mediating neurotransmission. Therefore, at least for botulinumtoxin, its internalization in this area and the subsequent translocationof the light chain from the endosome places the light chain in closeproximity to the regulated SNARE proteins. One reason that clostridialneurotoxins preferentially bind to this area is that the presynapticmembrane contains very high concentrations of ganglioside GT-1. However,GT-1 ganglioside or others that have slightly less affinity are presentalong the entire length of peripheral axons, albeit at much lowerconcentrations than at the presynaptic membrane. It has been shown thatboth botulinum and tetanus toxin bind to these regions but are notinternalized. See e.g., Angelis M. V. et al., Anti-GD1a Antibodies FromAn Acute Motor Axonal Neuropathy Patient Selectively Bind To Motor NerveFiber Nodes Of Ranvier Journal of Neuroimmunology 121 (2001) 79-82,hereby incorporated herein by reference. However, if the boundclostridial neurotoxins are exposed to high acidity they wouldtranslocate their light chains into the axons.

Neurons form their proteins at the cell body and transport them throughthe axon the entire distance from the axon to the synapse. It is knownthat among the proteins transported in this manner are the regulatedSNARE proteins. See e.g., Diefenbach R. J. et al., The Heavy Chain OfConventional Kinesin Interacts With The Snare Proteins SNAP25 AndSNAP23, 41 BIOCHEMISTRY 14906-14915 (2002), hereby incorporated hereinby reference. Therefore translocation of clostridial neurotoxin lightchains, at any point along the axon, allows them to cleave thetransported regulated SNARES thereby rendering them inactive. In manyclinical situations the tissue that would benefit from injection of aregulated SNARE inhibitor contains other nerve elements or cells thatperform regulated SNARE exocytosis. Therefore it is preferable that ifthe nerve in need of inhibition can be easily accessed at any pointalong its route, it can be selectively inhibited. As an illustrativeexample, a patient has a cancer involving a bone of the left foot and isin need of analgesia. Administration of a regulated SNARE inhibitor canbe done in and around the bone cancer to block pain fibers however, anundesirable side effect is that nerves to muscles in the area are alsoinhibited and the muscles are temporarily paralyzed. Although motor andsensory nerve fibers are mixed in peripheral nerves they separate priorto entering into the spinal cord. Specifically, motor fibers enter moreanterior than sensory fibers, they divide into the ventral and dorsalroots of the spinal nerve from that segment of spinal cord. At thedorsal root the sensory fibers can be accessed and blocked withouteffecting the motor nerve, thereby avoiding paralysis. This shows howthe methods of this invention allow selective inhibition of nerves thatinnervate a tissue. Numerous other situations where this is of valuewould be evident to a person skilled in the art, and further detailedexamples are provided below.

In one embodiment of the acid mediated transport methods of theinvention, regulated SNARE inhibitors are injected from about one-secondto about two hours prior to acidification, thereby allowing regulatedSNARE inhibitors to bind prior to translocation. In another embodiment,injection of regulated SNARE inhibitors is performed simultaneously withinjection of an acid formulation, thereby causing more general andnon-specific binding. The injection of acid solution could also precedeinjection of the protein of the inventions.

In one embodiment, regulated SNARE inhibitors or formulations thereof,are administered locally by injecting according to standard techniques.Local injection of regulated SNARE inhibitors can be performed by needleinjection, needleless pressure injection, biodegradable andnon-degradable implants and implantable pumps. Further non-limitingexamples are to incorporate the SNARE inhibitors onto coatings or partsof intravascular stents, implanted artificial or transplanted organs ortissues.

For local injection, the compounds of the invention can be formulated inphysiologically compatible aqueous solutions, such as Hanks's solution,Ringer's solution, or physiological saline buffer.

Another embodiment of the invention is to inject the protein of theinvention into an enclosed space such as pleural cavity, joint spaces,gastrointestinal, genitourinary or reproductive organs, lymphatics, andblood vessels. Another embodiment of the invention is to inject theprotein of the invention into tissues or organs of relatively homogenouscell types. Such organs would include the central nervous system,peripheral nerves, endocrine glands, liver and pancreas, bone marrow,cartilage, connective tissue, fat, nasal cavity and nasal sinuses, andpathologic material such as benign and malignant tumors. In anotherembodiment of the invention, the toxins of the invention are injectedinto vessels supplying specific areas, lymphatics, cerebrospinal fluid,anterior or posterior chamber of the eye, the cochlea or middle ear,synovial or pleural cavities.

7.1.2 Topical Administration

As used herein, the term “topical administration” or “topical delivery”means intradermal administration of a regulated SNARE inhibitor byadministration of the regulated SNARE inhibitor or a compositioncomprising the regulated SNARE inhibitor to intact skin. For example, byrubbing a composition of the invention onto an area of intact skin or byplacing an intradermal patch comprising a composition of the inventiononto an area of intact skin. The term “topical composition” means apharmaceutical composition designed for topical administration andcontaining a pharmaceutical.

Regulated SNARE inhibitors can be applied topically in intradermal ortransdermal formulations, such as solutions, creams, emollients,ointments, patches, aerosols, sprays, and mists. Processes to spreaddiffusion across mucosal or skin barriers can be used, for example byuse of liposomes or iontophoresis. One embodiment of the invention is toadminister regulated SNARE inhibitors in topical, slow-release solids,gels or polymers by application to the skin or mucus membranes. Specificembodiments include biodegradable contact lens, a coated orbiodegradable implant or stent for the nose, sinuses, Eustachian tube orin the form of a myringotomy tube, intrauterine device, or indwellingcatheter. Further examples are obvious to those skilled in the art.

7.1.3 Activation of Amphipathic Proteins or Their Conjugates

According to one embodiment of the invention, regulated SNARE inhibitorscan be used in an inactive or wild-type form. In another embodiment,regulated SNARE inhibitors can be used in a form that is activated atbody temperature upon exposure to typical in vivo conditions or thespecific local conditions found in specific tissues. In still anotherembodiment, regulated SNARE inhibitors can also be designed such thatunder certain conditions they are activated. For example, activation byexternal signal or external or internal energy sources such aselectrical current, microwave, ultrasound, or hypertherma orhypothermia.

7.2 Dosages

Pharmaceutical preparations suitable for use with the present inventioninclude compositions wherein the regulated SNARE inhibitors are presentin effective amounts, i.e., in amounts effective to achieve the intendedpurpose, for example, modulation of cellular function or treatment ofdisease. Of course, the actual amounts of the regulated SNARE inhibitorseffective for a particular application will depend upon a variety offactors including, inter alia, the disease being treated, the age andweight of the subject and, where appropriate, the judgment of theprescribing physician. Determination of effective amounts is well withinthe capabilities of those skilled in the art, especially in light of thedetailed disclosure herein.

The regulated SNARE inhibitors can be locally administered in any mannerthat achieves the requisite therapeutic or prophylactic effect.Therapeutically or prophylactically effective doses of the regulatedSNARE inhibitors can be determined from animal or human data. Theapplied doses can be adjusted based on the relative bioavailability,potency and in vivo half-life of the administered regulated SNAREinhibitors. Adjusting the dose to achieve maximal efficacy in humansbased on methods that are well-known is well within the capabilities ofthe ordinarily skilled artisan.

Typically, dosages and therapeutically effective amounts range fromabout 0.001 units to about 10,000 units of the regulated SNAREinhibitor, preferably, from about 0.1 units to about 1000 units, morepreferably, in the range of about 1 units to about 100 units. Typically,each dose is administered once a day to once every 3 years, preferably,once a week to once a year, more preferably once a month to once a year.

8. Treatment of Disease Using the Methods of the Invention to DeliverRegulated Snare Inhibitors

The methods, compositions, and formulations of the invention are usefulfor treatment and/or prevention of disease in mammals.

The methods, compositions, and formulations are useful to treat alldisease, conditions, and symptoms disclosed in WO 02/00172, to IraSanders, PCT/US01/20523(published Jan. 3, 2002), which is herebyincorporated herein by reference, and the methods and formulationsdescribed therein can be adapted in the present invention.

In one embodiment, the methods of the invention are directed treatment,reduction of symptoms, and/or prevention of disease in mammals usingacid mediated translocation of regulated SNARE inhibitors. In anotherembodiment, the methods of the invention are directed to treatment,reduction of symptoms, and/or prevention of disease in mammals bytranslocation and binding of regulated SNARE inhibitors by way ofprotein transduction domains. In yet another embodiment, the methods ofthe invention are directed to treatment, reduction of symptoms, and/orprevention of disease in mammals by translocation and binding ofregulated SNARE inhibitors by way of encapsulation vectors.

The methods of the invention are useful for the treatment, reduction ofsymptoms, and/or prevention of achalasia, anal fissure, anismus,blepharospasm, cerebral palsy, cervical dystonia, cervicogenic headache,hemifacial spasm, dyshidrotic eczema, dysphagia, dysphonia, esophagealdysmotility, esophageal muscular ring, esotropia (infantile), eyelift,facial myokemia, gait disturbances (idiopathic toe-walking), generalizeddystonia, hemifacial spasm, hyperfunctional facial lines (glabellar,forehead, crow feet, down-turned angles of the mouth), hyperhidrosis,incontinence (spinal cord injury), migraine headache, myoclonus,myofascial pain syndrome, obstructive urinary symptoms, pancreas divisumpancreatitis, Parkinson's disease, puborectalis syndrome, reduction ofsurgical scar tension, salivary hypersecretion, sialocele, sixth nervepalsy, spasticity, speech/voice disorders, strabismus, surgery adjunct(ophthalmic), tardive dyskinesia, temporomandibular joint disorders,tension headache, thoracic outlet syndrome, torsion dystonia,torticolis, Tourette's syndrome, tremor, whiplash-associated neck pain,pain, itching, inflammation, allergy, cancer and benign tumors, fever,obesity, infectious diseases, viral and bacterial, hypertension, cardiacarrhythmias, vasospasm, atherosclerosis, endothelial hyperplasia, venousthrombosis, varicose veins, apthous stomatitis, hypersalivation,temporomandibular joint syndrome, sweating, body odor, acne, rosacea,hyperpigmention, hypertrophic scars, keloid, calluses and corns, skinwrinkling, excessive sebum production, psoriasis, dermatitis, allergicrhinitis, nasal congestion, post nasal drip, sneezing, ear wax, serousand suppurative otitis media, tonsil and adenoid hypertrophy, tinnitus,dizziness, vertigo, hoarseness, cough, sleep apnea, snoring, glaucoma,conjunctivitis, uveitis, strabismus, Grave's disease, asthma,bronchitis, emphysema, mucus production, pleuritis, coagulationdisorders, myeloproliferative disorders, disorders involvingeosinophils, neutrophils, macrophages and lymphocytes, immune toleranceand transplantation, autoimmune disorders, dysphagia, acid reflux,hiatal hernia, gastritis and hyperacidity, diarrhea and constipation,hemorrhoids, urinary incontinence, prostatic hypertrophy, erectiledysfunction, priapism and Peyronie's disease, epididymitis,contraception, menstrual cramps, preventing premature delivery,endometriosis and fibroids, arthritis, osteoarthritis, rheumatoid,bursitis, tendonitis, tenosynovitis, fibromyalgia, seizure disorders,cerebral palsy, spasticity, headache, and neuralgias.

In a preferred embodiment, the methods of the invention are useful forthe treatment, reduction of symptoms, and/or prevention of pain orinflammation; migraine headaches; allergy; cystic fibrosis; diseaserelated to adipose tissue; viral infection; cancer; fever; sweating,eccrine, and apocrine; disease related to or associated with holocrinesecretions and acne; disease relate to mucous secretion; prostatichypertrophy; diseases treatable by gene therapy; disease of the veins,such as venous stasis, varicose veins and hemorrhoids; high bloodpressure. The methods of the invention are also directed to use ofregulated SNARE inhibitors simultaneously as a therapeutic agent and asa vaccine.

In another embodiment, the invention is directed to preparation of amedicament or pharmaceutically acceptable formulation, preferably amedicament or pharmaceutical acceptable formulation suitable for localdelivery, comprising a therapeutically effective amount of a regulatedSNARE inhibitor for use in the methods of the invention.

8.1. Pain And Inflammation

8.1.1 Pain

In one embodiment of the invention, therapeutically effective amounts ofregulated SNARE inhibitors are administered locally, preferably, by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors—so as to increase or facilitate cell membranetranslocation and/or binding—to treat, reduce the symptoms of, and/orprevent pain in mammals, and the related process of inflammation, whichcauses pain.

Pain is a noxious sensation mediated by a special class of neuronscalled nociceptors. The majority of these nociceptors are unmyelinated Cfibers and the thin myelinated A delta fibers, which are concentrated inthe skin and mucosa. Nociceptors have membrane receptors that respond toheat, acid, and a wide variety of endogenous bioactive substancessecreted by other neurons surrounding tissue cells and from white bloodcells that migrate out of the blood stream to the involved area.

Unmyelinated C fibers are unusual in that the terminal axons of singleneuron cover a relatively wide area, up to several centimeters in skin.Stimulation of a single terminal is conveyed directly to the otherterminal axons of the same neuron as well as sent back to the CNS. Thedirect transmission of signals from one terminal axon to another iscalled axon-axonal reflex and plays an important role. These Cnociceptors have the ability to release neuropeptides thereby causing aninflammatory reaction in surrounding areas not involved in the originalpainful stimuli. Neuropeptides released by nociceptors include substanceP and CGRP. Substance P has numerous effects, it can stimulate painsensation in other nociceptors and activate local tissue cells calledmast cells that contain a wide variety of bioactive substances thatcontribute to inflammation and further pain. CGRP is a powerful dilatorof blood vessels and causes increased circulation to the area. Theresultant reaction amplifies the original noxious insult by neuralmechanism and by activation of surrounding cells. The clinical signs ofinflammation have been described for centuries as heat (due to increasedcirculation), swelling (secondary to fluid passing from local bloodvessels into the tissue), redness (due to the increased bloodcirculation), and pain (noxious sensation from the original insult aswell as the numerous bioactive substances released in the area).

Sensory neurons have their cell bodies in ganglia outside the centralnervous system and have two axons. One axon courses down peripheralnerves to be distributed to innervate tissue, and the second axon passesto the spinal cord. Activation of the membrane receptors of the terminalaxons of the nociceptors initiates a neural signal that passes along thelength of the neuron to secondary relay neurons in the spinal cord andbrainstem. The principal neurotransmitters at the synapses betweennociceptor neuron and the relay neuron are substance P, glutamate, CGRPand neuropeptide Y. Some processing of the signal occurs at this synapsethat either inhibits or enhances the signal. In addition, under certainconditions the signal can elicit local reflexes at the spinal-cord levelthat are sent through efferent neurons to the periphery tissue, furtheramplifying the reaction.

As pain is a symptom of many different diseases and perhaps the singlelargest cause of human suffering it has been a priority for therapeuticintervention. Therapeutic interventions have been devised to interveneat many points from the peripheral tissue to brain. Examples oftreatments for treating pain at the tissue level include steroids andnon-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin; and inthe CNS the principle drugs used are narcotics such as Demerol. Aspirinblocks production of prostaglandins, an important mediator of pain andinflammation produced by tissue cells. Aspirin is effective for mildpain from some sources but does relief strong pain. Moreover, aspirin isassociated with significant side effects, it inhibits blood clotting,thereby making the patient susceptible to spontaneous bleeding, itirritates the gastrointestinal tract and can cause gastritis and ulcers,and chronic use can cause tinnitus. Narcotics are the primary treatmentfor severe pain. Narcotics work by inhibiting the transmission of painsignals in the CNS. However, narcotics have numerous side effects:respiratory depression; clouded sensorium; and addiction. In practicethe exaggerated fear of addiction causes physicians to routinelyunder-dose patients, and this is a major cause of suffering in thepatient population most in need of pain relief.

In their wild type form, the Clostridia tetani have some analgesic andanti-inflammatoryproperties with tetanus toxin having more effects thenbotulinum toxin. In clinical practice, wounds infected solely byClostridia tetani are not painful. In fact, some patients are unawarethey have an infection until the onset of tetanic symptoms. Tetanustoxin is known to bind to and be internalized by all nerves includingsensory nerves such as the nociceptors. Finally, another importantdifference between tetanus toxin and all the botulinum toxin serotypesis that tetanus toxin can enter into and inhibit white blood cells suchas macrophages that are drawn to injured tissue and greatly amplify theinflammatory response. For these reasons, tetanus neurotoxin is moreversatile then botulinum toxin as an analgesic or anti inflammatoryagent, although it is not ideal as most of the toxin is transported bynerves out of the area. WO 02/00172: Methods for using tetanus toxin forbeneficial purposes in animals (mammals), hereby incorporated herein byreference teaches the use of tetanus toxin for the treatment of pain andinflammatory conditions.

It is believed that botulinum toxin has a significant effect on painthat is caused by muscle spasms, this being secondary to the inhibitionof muscle contraction. In addition, injections of botulinum toxin intothe temporalis and frontalis muscles has been shown to be effective inthe treatment of headache (Binder W. et al., (2000) Botulinum toxin typeA (Botox®) for the treatment of migraine headache, Otolaryngol Head andNeck Surg 123: 169-176, hereby incorporated herein by reference).However, based on animal experiments it has also been claimed thatbotulinum toxin has an effect on inflammatory evoked pain (U.S. Pat. No.6,063,768: Application of botulinum toxin to the management ofneurogenic inflammatory disorders), and chronic pain syndromes (U.S.2004/0028706A1: Neuralgia pain treatment by peripheral administration ofa neurotoxin), hereby incorporated herein by reference. Borodic teachesagainst the use of botulinum toxin due to paralysis of musclessurrounding the injection site, see e.g., U.S. Pat. No. 6,429,189:Cytotoxin (Non-Neurotoxin) For The Treatment Of Human Headache DisordersAnd Inflammatory Diseases, hereby incorporated herein by reference).Moreover, controlled experiments in humans fail to show any directeffect of botulinum toxin on pain (Blersch W. et al., (2002) BotulinumToxin A And The Cutaneous Nociception In Humans: A Prospective,Double-Blind, Placebo-Controlled, Randomized Study, J. Neurol. Sci.,205:59-63), hereby incorporated herein by reference. In fact there arereceptors for acetylcholine on nociceptors, and stimulation of thesereceptors decreases pain (Dusser G, et al., Cholinergic Modulation OfNociceptive Responses In Vivo And Neuropeptide Release In Vitro At TheLevel Of The Primary Sensory Neuron, Pain 107 (2004) 22-32), herebyincorporated herein by reference.

Due to the shortcomings of wild type Clostridia tetani investigatorshave developed modified forms of the toxins that bind specifically tosensory neurons. to nociceptors U.S. Pat. No. 5,989,545: ClostridialToxin Derivatives Able To Modify Peripheral Sensory Afferent Functions,hereby incorporated herein by reference, discloses a modified botulinumtoxin in which the binding domain is replaced by a compound that bindsto sensory neurons. Therefore alight chain is introduced to nociceptorneurons for the purposes of blocking the synapse between the nociceptorneuron and the first relay neuron in the CNS. WO/0057897A1: Use Of ALectin Or Conjugates For Modulation Of C-Fibre Activity, herebyincorporated herein by reference, specifically discloses the use of alectin to replace the wild type binding domain, as lectins have specificaffinity for the nociceptor c fibers. In U.S. 2003/0165541A1: Methodsfor treating inflammation pain, hereby incorporated herein by reference,the specific target of the toxin is changed from a neuron to anon-neuronal cell that contains receptors for substance P. Although manydifferent cell types contain substance P receptors, those contributingto the inflammatory response are mast cells and endothelial cells. TypeTC mast cells (those containing the enzymes trypsin and chymase) arefilled with large vesicles containing many inflammatory bioactivesubstances. When properly stimulated they release all these substancesin a rapid process called degranulation. However, this degranulationdoes not utilize regulated SNAREs and therefore this function would notbe impaired by CNT.

Preventing the vesicle mediated replacement of cell membrane at thesensory endings of the nociceptors prevents the replacement ofnociceptors membrane receptors and cause their depletion and the neuronstops responding to pain. Stopping neurotransmitter release at thesynapse between the primary and projection neuron blocks thetransmission of pain. Furthermore, any process that interferes with theaction potentials propagating along the axon will also block painsignals.

According to the invention, pain is blocked at various points along thepathway described above by locally administering regulated SNAREinhibitors according to the methods of the invention to effect efficientcell membrane binding and/or translocation.

In one example of this embodiment, a male patient experiences pain inthe elbow region. To alleviate this pain a needle is passed through theskin above the elbow and small continuous pulses of electricity areapplied through the tip of the needle. When the needle tip touches thenerve supplying the area from which pain is experienced he feels atingling in the area that the nerve distributes to, and informs theclinician. The clinician advances the needle slightly until the needletip is under the perineurium of the nerve. The clinician slowly infuses5 ml of normal saline containing 10 units of tetanus neurotoxin. Theclinician waits for 2 minutes to allow the solution to diffuse along thenerve and for the tetanus neurotoxin to bind. As the pain neurons aremostly unmyelinated C fibers, their entire axonal membrane is exposedfor the tetanus neurotoxin to bind. In contrast, the myelinated fibersare covered by Schwann cells and only a small percentage of theirmembrane is exposed at the nodes of Ranvier. After the waiting period, acontinuous current of 1 amp is passed through the needle tip in a pulselasting 10 seconds. A pH below 6.0 is generated in the region of thenerve around the needle tip. This causes the tetanus neurotoxin topreferentially translocate its light chains into the neurons. Theselight chains then diffuse proximal and distal over the next 24 hours toreach presynaptic membranes where they block vesicle release, therebyblocking pain sensation.

As a further example of blocking pain, botulinum neurotoxin C2 can betranslocated across axon membranes to stop the propagation of axonaction potentials. Botulinum neurotoxin C2 exists as separate heavy andlight chains that bind at the cell surface and are internalized intoendosomes. After acidification, the C2 light chain translocates into thecytoplasm. C2 disassembles actin, therefore, disrupting the cytoskeletonand transport. Injection of Acid/C2 under the epineural of a peripheralnerve would establish conditions for translocation of the C2 light chaininto axons; disruption of tubules causes a local area where actionpotentials do not propagate thereby blocking pain sensation.

As a further example, a 40-year-old female has rheumatoid arthritis withsevere left knee pain. The clinician injects the serosal joint cavitywith a solution of 30 units of botulinum neurotoxin dissociated lightand heavy chains with anthrax protective antigen (the amphipathiccomponent of anthrax toxin) in an acidic carrier solution. The botulinumneurotoxin light chains are thereby translocated into the nociceptiveneurons and cause them to stop recycling membrane receptors therebyrelieving the pain.

-   -   8.1.1.1 Pain From Migraine Headaches

In yet another embodiment of the invention, therapeutically effectiveamounts of regulated SNARE inhibitors are administered locally,preferably, by way of acid mediated translocation, protein transductiondomains, or encapsulation vectors—so as to increase or facilitate cellmembrane translocation and/or binding—to treat, reduce the symptoms of,and/or prevent the pain associated with migraine headaches in mammals

It is thought that the etiology of migraine and other headaches is aneurogenic allergic reaction. The initial brief vasoconstriction isfollowed by a vasodilation. The vasoconstriction causes the aura, abrief neurological aberration. The following vasodilation is painful. Ithas been shown that neural activity alone can cause mast celldegranulation around the dura. The nerve supply of the cerebral vesselsthrough autonomic neurons in the sphenopalatine ganglion, these join thecarotid arteries when they pas through the skull base. Some treatmentsof migraine use ergotamines, a substance that causes prolongedvasoconstriction. Blocking the neurogenic pathway can treat themigraine.

In one example of this embodiment of the invention, a 30-year-old femalewith recurrent left-sided migraine headaches is injected with 10 unitsof botulinum neurotoxin A into her pterygopalatine space by passing a 27gauge needle 1.5 inches through the sphenopalatine canal and injectingthe botulinum neurotoxin in 3 ml of saline carrier solution. Patientreports a resolution of her migraine for 3 months.

8.1.2 Inflammation And Associated Pain

In one more embodiment of the invention, therapeutically effectiveamounts of regulated SNARE inhibitors are administered locally,preferably, by way of acid mediated translocation, protein transductiondomains, or encapsulation vectors—so as to increase or facilitate cellmembrane translocation and/or binding—to treat, reduce the symptoms of,and/or prevent inflammation and the pain associated with inflammation inmammals.

Regulated SNARE inhibitors are useful to treat inflammation. Forexample, U.S. Pat. No. 6,063,768, hereby incorporated herein byreference, discloses application of botulinum toxin to manage neurogenicinflammatory disorders, especially in rheumatoid joints, which is herebyincorporated by reference herein. However, such methods suffer from lackof efficiency of cell membrane translocation and/or binding.

In one example of this embodiment of the invention, a 60 year old femalehas severe rheumatoid inflammation in her right knee. She undergoesinjection of 20 units of anthrax protective antigen/lethal toxin in 2 mlnormal saline into the knee. The following day the inflammation hassubsided.

8.2 Allergy

In one embodiment of the invention, therapeutically effective amounts ofregulated SNARE inhibitors are administered locally, preferably, by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors—so as to increase or facilitate cell membranetranslocation and/or binding—to treat, reduce the symptoms of, and/orprevent allergy in mammals.

The methods of the invention are useful to treat, for example, allergicconditions, such as allergy related rhinitis, asthma, conjunctivitis,gastroenteritis, serous otitis, sinusitis and dermatitis, and relatedconditions, such as infectious sinusitis and otitis media that occursecondary to allergy induced mucosal swelling. According to the methodsof the invention, regulated SNARE inhibitors are administered to thebody structure and/or the nerves and nerve ganglia supplying thesestructures. In the case of botulinum neurotoxin C2 the target is not theneuron but the mast cell.

Common to allergies is the involvement of the IgE class of antibody.Individuals are not born with allergies, rather, they acquire them byexposure to allergens. The steps of the IgE allergic reaction aresensitization upon first exposure to the allergen, and then the allergicresponse to subsequent exposures. The allergic response consists of animmediate and delayed response referred to as the early and late phaseresponses respectively. In atopic individuals, those prone to allergies,the initial exposure to an antigen results in the production of IgEantibodies that specifically recognize that allergen. This process iscalled sensitization.

The early-phase response (ERP) is the immediate reaction that occurswithin minutes of exposure to an allergen. IgE are bound to the surfaceof a neuroimmune cell called the mast cell (in the circulation thesecells are called basophils). Sufficient numbers of bound IgE antibodiesthat react with an allergen causes the mast cell to release contetanusneurotoxin of secretory vesicles, a process known as degranulation. Thesecretory vesicles contain histamine and other stored substances such asnerve growth factor (NGF). In addition, the mast cell and T cellsimmediately begin manufacturing leukotrienes, cytokines, enzymes andsubstances that activate blood platelets and attract secondary cells tothe area. Eosinophils produce major basic protein, eosinophil cationicprotein, leukotrienes and nerve growth factor. TH2 lymphocytes releasecytokines that promote further IgE production and eosinophil chemoattraction, and increased numbers of mast cells. The sensory nervestimulation causes reflexes that are designed to aid in defending thetissue. These reflexes are often a larger problem then the localallergic response. Reflexes can range from large gross motor actions toregional afferent and efferent arcs or even local axon-axonal reflexesinvolving a single neuron.

Some reflexes recruit major motor actions that are well recognized. Inthe nose sneezing is a reflex attempt to expel unwanted material andcoughing is the equivalent response in the lungs.

Regional reflex arcs involve the sensing of the stimulus by the sensoryneuron, the transfer of the message to the ganglia and the centralnervous system and an efferent response via autonomic neurons. Reflexexcitation by the autonomic nervous system directly causes mast cell todegranulate, thereby spreading the reaction. In addition, these reflexescontrol a variety of other functions. In the nose, these reflexes causeincreased mucus production, increased cilia movement, and congestion. Inthe lungs, reflexes cause bronchospasm, increased mucosal congestion,and production of airway secretions. In the GI tract, reflexes causedysmotility, mucosal congestion, and secretions. In the skin, thereflexes cause swelling and itching.

Finally, there are local axon-axonal reflexes in sensory nociceptivenerve fibers. Allergic stimulation of a single neuron causes release ofmediators from other axons of the same neuron.

In chronic allergic stimulation, the mast cells and eosinophils releasesnerve growth factor that causes growth of the nerves in the region.Thereby allowing for increased neural responses and hyper reactivity.

There is a great need for an effective treatment for allergic disorders.It has long been thought that the allergic reaction involved onlyhistamine release by mast cells. Therefore, first line therapy forallergy was antihistamines, or more recently the non-sedatingantihistamines. Other therapies are directed to block the effects of themast cell secretions with adrenergic agonists. It is not obvious tothose skilled in the art that a central role in allergic disordersinvolves the autonomic nervous system and that this nerve activity canbe blocked by regulated SNARE inhibitors for a beneficial effect.

In one embodiment of this aspect of the invention, upon localadministration, regulated SNARE inhibitors interfere with the allergicprocess by: (1) Directly blocking degranulation by the mast cell(principally botulinum neurotoxin C2), (2) Block the degranulation ofthe mast cell induced by autonomic nerve activity. (3) Decrease humoralrelease during axonal reflexes. (4) Decrease the parasympatheticeffector arm of reflex allergic responses (5) Decrease the increasedtonic activity of the autonomic systemic that is related to priorallergic reactions. (6) Decrease the nerve enlargement induced by nervegrowth factor released during allergic reactions. (7) Reverse certaincomplications of allergic reactions such as mucosal thickening bydecreasing autonomic nerve activity. (8) Combinations of botulinumneurotoxin and tetanus neurotoxin can have a synergistic effect: forexample, botulinum neurotoxin can block parasympathetic nerves whiletetanus neurotoxin can excite sympathetic nerves thereby causingdecongestion.

In one example of this embodiment of the invention, a 30-year-old malehas seasonal allergic rhinitis. In May, prior to pollen formation, hehas 30 units of botulinum neurotoxin topically applied in each nostril.Specifically, the botulinum neurotoxin is absorbed onto cotton pledgetsthat are placed into each nasal cavity for one hour. In the followingmonths, the symptoms he normally experiences, itching, sneezing andnasal congestion are significantly reduced. Alternatively, the samepatient can be treated with 20 units of botulinum neurotoxin and 10units of tetanus neurotoxin to combine an anti-parasympathetic effectwith a sympathomimetic effect. Alternatively, if decongestion isdesired, 10 units of tetanus neurotoxin can be topically applied. Inanother embodiment, 1 unit of recombinant DNA coding for tetanusneurotoxin is pressure injected across the nasal mucosa to transfectmucosal cells. These cells then express the tetanus neurotoxin formonths.

In another embodiment the light chains from 30 units of botulinumneurotoxin/E in 10 ml saline are sprayed into each nostril by anatomizer. A thin electrolytic membrane is placed over the mucusmembranes of each inferior turbinate. Electrical current is appliedacross the biogel and its mucosal side becomes acidic thereby allowingtranslocation of the botulinum neurotoxin/E light chins into mucosa,mast cells and neurons.

8.3 Cystic Fibrosis

In yet another embodiment of the invention, therapeutically effectiveamounts of regulated SNARE inhibitors are administered locally,preferably, by way of acid mediated translocation, protein transductiondomains, or encapsulation vectors—so as to increase or facilitate cellmembrane translocation and/or binding—to treat, reduce the symptoms of,and/or prevent cystic fibrosis and related conditions.

Regulated SNARE inhibitors can be incorporated into cell membranes tocontrol the interaction of the cell with its environment, for example,transporter and signal transduction membrane proteins, identifyingantigens, and others. An example of a disease that can be treated usingthis embodiment of the invention is cystic fibrosis cystic fibrosis is acondition in which a membrane transport protein is missing inrespiratory epithelial mucosal cells. As a result, the respiratorysecretions are excessively thick. The conjugate to the amphipathicproteins might be the missing transport protein.

8.4 Disease Related To Adipose Tissue

In still yet another embodiment of the invention, therapeuticallyeffective amounts of regulated SNARE inhibitors are administeredlocally, preferably, by way of acid mediated translocation, proteintransduction domains, or encapsulation vectors—so as to increase orfacilitate cell membrane translocation and/or binding—to treat, reducethe symptoms of, and/or prevent disease related to adipose tissue inmammals.

Increased adipose tissue is a major health problem in industrializedsocieties. Decreased caloric intake is associated with fewer chronicdiseases, such as diabetes and hypertension, as well as a longer lifespan. Also, the reduction of or repositioning of fat deposits isdesirable for cosmetic reasons. In order for adipocytes to take upglucose from the circulation, cell vesicles containing the glucosetransporters (GLUT) add the enzyme to cell membrane. The increaseduptake of glucose is converted into fat. Tomori et al. showed thatbotulinum neurotoxin could prevent the docking of these vesicles inadipocytes permeabilized with streptolysin-O. Chen demonstrated thatadipocytes had SNAP-23, a SNAP isoform that is not cleaved by botulinumneurotoxin-A, however VAMP are involved in this process and they can becleaved by botulinum neurotoxin-B introduced by incubation in low ionicstrength medium.

According to the invention, obesity can be treated by inducing the lightchains of clostridial neurotoxin to translocate through adipose cellmembranes when the extracellular fluid is acidified (clostridialneurotoxin cannot penetrate adipose membranes under normal extracellularconditions).

In one example of this embodiment, about 5 ml of normal salinecontaining the light chains from 50 units of botulinum neurotoxin isinjected into the abdominal adipose tissue of a 30 year old women. Astimulating electrode is passed into the target area and electricalstimulation is applied as the needle is passed multiple times parallelto the skin, about 1 cm deep. The electrical stimulation causes andacidic environment at its tip. In this manner, the adipocytes that wereadjacent to the needle tip throughout its movements experience amomentary acidification, thereby effecting translocation of thebotulinum neurotoxin light chains. After entering the adipose cells, thelight chains block vesicular activity thereby preventing significantglucose uptake. The adipocytes metabolize their own fat stores andslowly shrink in size. The result is a shrinking of the volume ofadipose tissue in the area and a enhanced cosmetic appearance. The useof lytic toxins from anthrax, diptheria, and others can be used to causelysis of the cells and remove them permanently.

The sympathetic system controls fat metabolism in adipocytes to someextent, perhaps through special receptors such the beta 3 receptor.Thus, another example of this embodiment of the invention. Injection oftetanus neurotoxin raises the sympathetic tone and promote utilizationof lipid stores. Therefore, injection of 10 units of tetanus neurotoxinin 5 ml of saline into the subcutaneous abdominal fat causes it todecrease in size even without acid mediated translocation.

8.5 Viral Infection

In yet another embodiment of the invention, therapeutically effectiveamounts of regulated SNARE inhibitors are administered locally,preferably, by way of acid mediated translocation, protein transductiondomains, or encapsulation vectors—so as to increase or facilitate cellmembrane translocation and/or binding—to treat, reduce the symptoms of,and/or prevent viral infections in mammals.

Viral infections can be treated with regulated SNARE inhibitors, forexample, an upper respiratory tract infection or “common cold”. Viralinfection of the nasal mucosa causes inflammation manifested ascongestion, primary and neurogenic inflammation. These cases last amatter of days, and according to the invention, can be treated with thebotulinum neurotoxin E or its light chain, or its light chain in aamphipathic protein conjugate with the botulinum neurotoxin C2II.

Botulinum neurotoxins C2 is not a neurotoxin, it is composed of 2chains, C2II is the larger chain and contains the binding andtranslocating domains. C2I is the toxic chain that exhibitsdepolymerizing action. Actin is needed for cellular skeleton movementand secretion of certain vesicles. C2II binds to cells and then isjoined by C2I, which begins the endosomal internalization stage. Theessential part of C2I that allows it to bind to C2II are the first 250amino acids of its N-terminal. If these same binding fragments arecovalently bound to another toxin, it will also be translocated intocells by the C2II. Among the cells that C2 binds with are the mastcells, which mediate much of the inflammatory and allergic reactionswithin nasal mucosa. For example, see U.S. Pat. No. 6,429,189 (issuedAug. 10, 2002), hereby incorporated herein by reference, which disclosescytotoxin (non-neurotoxin) for the treatment of human headache disordersand inflammatory diseases.

In one example of this embodiment of the invention, a 50 year old manwith a cold is treated by spraying into each nasal cavity 30 units ofbotulinum neurotoxin C2II/E hybrid. The congestion and excessive mucoidproduction improve for the following 3 days.

In another example, the same patient is treated with 20 units ofbotulinum neurotoxin B in 2 ml saline placed on cotton pledgets, whichare placed onto each turbinate for one hour.

8.6 Cancer

In one embodiment of the invention, therapeutically effective amounts ofregulated SNARE inhibitors are administered locally, preferably by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors—so as to increase or facilitate cell membranetranslocation and/or binding—to treat, reduce the symptoms of, and/orprevent cancer in mammals.

The methods of the invention are useful for benign or malignant tumors,sarcomas (originating from mesenchymal cells), carcinomas (originatingfrom epithelial cells), or mixed or compound tumors. Sarcomas includethose from connective, endothelial, hematopoietic and muscle cells. (fisquamous cell carcinoma, basal cell carcinoma, adenocarcinoma, bladdercancer, glioblastoma, gliomas, astrocytoma, oligodendroglioma, breast,neuroendocrine, cholangiosarcoma, colorectal, head and neck,hepatocellular, chronic lymphocytic leukemia, acute myeloid leukemia,non-small cell lung carcinoma, mesothelioma, non-Hodgkin's lymphoma,cutaneous B-lymphoma, cutaneous T-cell lymphoma, melanoma, multiplemyeloma, myeloproliferative disease, myelodysplastic syndromes, ovarian,pancreatic, prostatic, renal cell carcinoma, and soft tissue sarcoma.

Cancer involves inappropriate cell function, most importantly cellgrowth. Part of the disturbed metabolism of cancer cells is the loss ofcontrol over certain aspects of vesicle trafficking. Recent research hasidentified that many proteins associated with the control of regulatedexocytosis and/or vesicle fusion in general are expressed at abnormallevels,. Most importantly, some of these proteins are the SNARE isoformscleaved by regulated SNARE inhibitors. See e.g., A Fusion Inhibitor OnEndosomes, The Journal of Cell Biology (2003) 162:125-137, herebyincorporated herein by reference; Sun W et al., Hrs regulates earlyendosome fusion by inhibiting formation of an endosomal SNARE complex,The Journal of Cell Biology (2003) 162:125-137, hereby incorporatedherein by reference; Chan A et al. A Putative Link Between ExocytosisAnd Tumor Development, Cancer Cell (2002) 6:427-8, hereby incorporatedherein by reference; Palmer R et al., Induction Of BAIAP3 By The EWS-WT1chimeric Fusion Implicates Regulated Exocytosis In Tumorigenesis, CancerCell (2002) 6:497-505), hereby incorporated herein by reference.

Many pharmaceuticals can impair cancer cell growth but are unable to beused in vivo as they cannot be selectively targeted to cancer cells.However, the clostridial neurotoxins are unusual in that the particularSNARE isoforms that they neutralize are not essential for normal cellsurvival. Surprisingly, genetic experiments that selectively eliminateeither SNAP-25, (see e.g., Washbourne P et al., Genetic Ablation Of TheT-SNARE SNAP-25 Distinguishes Mechanisms Of Neuroexocytosis, NatureNeuroscience (2002) 5:19-26, hereby incorporated herein by reference),or VAMP-2, (Schoch S et al., SNARE Function Analyzed inSynaptobrevin/VAMP Knockout Mice, Science (2001) 294, herebyincorporated herein by reference) in mice come to term with normal brainand body morphology,. Of course the fetuses are still born because allneurotransmission is blocked, including the muscles of respiration.However, the fact that all cells grow and differentiate normally showsthat SNAP 25 and VAMP 2 are not critical for normal cell survival.

According to the invention, clostridial neurotoxins can impair cancercell growth by interfering with vesicle trafficking. While not wantingto be bound by any theory, it appears that the unregulated growth ofcancer requires inappropriate use of regulated exocytosis pathways.Therefore, interference with these pathways inhibits the growth and maycause cancer cell death. Therefore the use of regulated SNARE inhibitorseither alone or by combination of multiple types of may be of increasedbenefit in certain types of cancer. Specifically combination of SNAP-25inhibitors (botulinum neurotoxins/A, C, E and IgA protease) may be usedwith VAMP-2 inhibitors (botulinum neurotoxins/B, F, G, H, and tetanustoxin). Also according to the invention, if clostridial neurotoxins isintroduced, the cells in a local area of the body that contain cancercells, only the cancer cells will be impaired, although of course nervefunction in the area will also be blocked.

Botulinum type C1 has some special features that make it a particularlyuseful therapeutic for cancer. botulinum neurotoxin/C1 cleaves syntaxin1 which is one of the core proteins in the SNARE mechanism (See e.g.,Foran P et al., Botulinum Neurotoxin C1 Cleaves Both Syntaxin AndSnap-25 In Uintact And Permeabilized Chromaffin Cells: Correlation WithIts Blockade Of Catecholamine Release, Biochemistry (1996) 35:2630-2636,hereby incorporated herein by reference; Williamson et al.: Mol. Biol.Cell 6:61a, 1995, hereby incorporated herein by reference; J. Biol.Chem. 271:7694-7699, 1996, hereby incorporated herein by reference).However, recent work has shown that syntaxin 1 has a second role incells. Specifically, syntaxin 1 is necessary for cell division (Seee.g., Conner S & Wessel G M: Syntaxin Is Required for Cell DivisionMolecular Biology of the Cell, Vol. 10, 2735-2743, August 1999, Syntaxinand 25-kDa synaptosomal-associated protein: Differential effects ofbotulinum neurotoxins C1 and A on neuronal survival, hereby incorporatedby reference). In Drosophila embryos, syntaxin 1 is necessary forvesicle trafficking at an early stage of embryogenesis. Without syntaxin1 embryos stop growing at a very early stage as cell division isimpaired (Burgess R et al., The Synaptic Protein Syntaxin 1 Is Requiredfor Cellularization of Drosophila Embryos, The Journal of Cell Biology,Volume 138, Number 4, Aug. 25, 1997 861-875, hereby incorporated hereinby reference).

Normally, chemotherapeutic agents that stop cell division have extremelytoxic side effects, in fact this is the basis for many of the worst sideeffects of cancer chemotherapy. However, botulinum neurotoxin C1injected locally does not appear to have any deleterious effects inhumans (Eleopra R, Botulinum neurotoxin serotypes A and C do not affectmotor units survival in humans: an electrophysiological study by motorunits counting Clinical Neurophysiology 113 (2002) 1258-1264,herebyincorporated herein by reference). In fact, clinical trials of botulinumneurotoxin/C1 injection into muscle have been done for the purposes ofevaluating it as a substitute for botulinum neurotoxin/A and no toxicside effects have been reported (Eleopra R et al., Botulinum neurotoxinserotype C: a novel effective botulinum toxin therapy in human.Neuroscience Letters 224 (1997) 91-94), hereby incorporated herein byreference).

According to the invention, botulinum neurotoxin C1 stops cell divisionand is therefore be of great value in cancer therapy, but does not haveany toxic effects when injected locally into humans. Syntaxin 1 fragment(which is cleaved by botulinum toxin C1) is one of the regulated SNAREproteins. But is has been very recently discovered that syntaxin 1 isnecessary for cell division. Within muscle tissue there no toxic effectdue to cell division inhibition because muscle cells are mature, butcancer is a disease where cell division is out of control. Thus,inhibitors of syntaxin 1, such as botulinum toxin C1 are useful to treatcancer.

Clostridial neurotoxins normally do not enter into non-neural cellstherefore it is unclear how they could be used as therapeutic agents.However, this invention discloses multiple methods of introducingproteins into cells. According to the invention, clostridial neurotoxinsand other regulated SNARE inhibitors can be delivered to all cells in atissue area with technology that has been well known in the art.Therefore, this embodiment of the invention combines a fortuitouscombination of disparate elements to provide a method of treatingcancer.

In one example of this aspect of the invention, a forty-year-old femalehas squamous cell carcinoma of the lung. A balloon catheter is threadedthrough the femoral artery to the branch of the pulmonary arterysupplying the lung lobe containing the tumor. The balloon is inflatedthereby occluding the artery and stopping circulation. One cc of asaline solution containing 20 units of wild type botulinum toxin C1 areinfused in conjunction with 10 cc of physiological solution buffered topH 4.5. The solutions thereby are delivered through out the distributionof the artery that includes the lung cancer. By the action of the acidicpH the light chains of botulinum C1 translocate into the cytoplasm ofthe cancer cells. After ten minutes slight suction is applied to thecatheter to retrieve any excess botulinum neurotoxin/C1 so that it doesnot spread systemically when blood flow is restored. The ballooncatheter is deflated and normal blood flow is restored. The catheter isremoved and the patient is observed overnight and then discharged fromthe hospital. Two weeks later she returns for a CT scan of the chest toevaluate the change in the tumor size.

In another embodiment of the above example, 20 units of botulinumneurotoxin/A are mixed with 20 units of botulinum neurotoxin/B. Inanother embodiment of the above example 40 units of Clostridia botulinumA are used.

In another embodiment of the above example, 100 units of Botulinum toxinlight chains are used in substitution for the whole toxin. The unitmeasure of light chain is the molar equivalent of a unit of whole toxin,i.e., the same number of molecules are present in each case. The pH fordirect translocation of light chains is lower than the whole toxin,preferably about pH 4.0. To ensure that in vivo tissue buffers do notinterfere with the method, a slightly lower buffered pH of 3.8 is usedin the toxin solution. In this example suction is not necessary toretrieve the light chains as they cannot enter any other cells in thebody and will be metabolized and excreted.

In another example, a fifty-year-old male with prostate cancer whoseprimary cancer has been resected has a metastatic lesion in the leftfemur. A long bone biopsy needle is placed through the skin and into themarrow of the femur and 10 ml of acidic solution buffered to pH 4.5 areinfused in conjunction with 20 units of botulinum neurotoxin A.

In another example a patient has a squamous cell carcinoma of the leftvocal fold. The patient is anesthetized and a laryngoscope is insertedto allow the clinician to directly visualize the tumor. One tenth of acc of viscous gel containing 100 units of botulinum C1 light chain isapplied onto the lesion.

8.7 Fever

In one embodiment of the invention, therapeutically effective amounts ofregulated SNARE inhibitors are administered locally, preferably, by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors—so as to increase or facilitate cell membranetranslocation and/or binding—to treat, reduce the symptoms of, and/orfever.

Abdominal processes that induce fever do so by way of the vagal branchto the liver. Sectioning that branch or chemodenervating it eliminatesfever. This is valuable in many clinical conditions, however, theproblem is selective delivery of drugs to the liver. The liver receivesall of the venous flow from the GI tract via the portal circulation. Oneof the areas that drain into this system is the venous system above theanal sphincter, known as the source for hemorrhoids.

According to this embodiment of the invention, a speculum or similardevice can be positioned in the anus so as to compress possiblecommunication of the portal venous system with the remaining systemicsystem. Clostridial neurotoxin is injected directly into this venousarea to be distributed to the liver.

In one example of this embodiment, a 30-year-old male with abdominalHodgkin's disease and fever undergoes an injection of 30 units ofbotulinum neurotoxin to the venous rectal area. The botulinum neurotoxinis circulated to the liver where it binds and blocks autonomic neuronsthereby stopping the signals for fever. Those skilled in the art canreadily appreciate that what is disclosed is a method of deliveringpharmaceuticals to the liver without significant systemic spread. Thissame technique can be used for a variety of pharmaceuticals and genetherapy agents.

8.8 Skin Disorders

Skin disorders are best discussed together as the barrier function ofthe skin is a special technical problem for drug delivery. The skincovers the external surface of the body and its outer layer, the stratumcorneum, is a non-viable water impermeable barrier composed of deadkeratinocytes. The only openings in this barrier are areas at which hairfollicles emerge, which simultaneously are where sebum glands arelocated, and the sweat ducts.

8.9 Sweating, Eccrine And Apocrine

In one embodiment of the invention, therapeutically effective amounts ofregulated SNARE inhibitors are administered locally, preferably, by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors—so as to increase or facilitate cell membranetranslocation and/or binding—to treat, reduce the symptoms of, and/orprevent conditions associated with eccrine and apocrine sweating.

Eccrine sweat glands are present throughout skin and produce the thin,watery secretion known as sweat. These glands are under cholinergiccontrol and are blocked by transcutaneous injections of either botulinumtoxin A or B. Injections in each axilla require approximately 100 unitsof botulinum toxin A or 2000 units of botulinum toxin B. With eithertoxin the decrease in sweating lasts four months or longer (Dressler Det al, Botulinum toxin type B for treatment of axillar hyperhidrosis JNeurol. 2002 Dec;249(12):1729-32). Apocrine sweat glands are mostlypresent in the axilla and genital area and produce a special sweat thathas lipid and protein components. In the presence of moisture fromeccrine glands, apocrine sweat is broken down by bacteria to produce abody odor. Botulinum neurotoxin injections into axilla block this bodyodor (see, e.g., Heckmann Met al., Amelioration of body odor afterintracutaneous axillary injection of botulinum toxin A. Arch Dermatol.2003 Jan. 139(1):57-9), hereby incorporated herein by reference. U.S.patent application publication 2002/0086036A1, hereby incorporatedherein by reference, teaches methods for treating hyperhidrosis and U.S.Pat. No. 5,766,605 (issued Jun. 16, 1998), hereby incorporated herein byreference, teaches treatment of autonomic nerve dysfunction withbotulinum toxin.

Despite the success of botulinum toxin injections a major drawback isthe need for multiple transcutaneous needle injections that areperceived as extremely painful. Therefore, there is a need for anon-injected, local delivery mechanism for the treatment ofhyperhidrosis.

In one example of this embodiment of the invention, a forty-year-oldfemale complains of excessive sweating and odor from her axilla. A warmmoist towel is placed over her axilla to open the sweat pores for tenminutes. Then 1 cc of a normal saline solution containing a regulatedSNARE inhibitor is placed over 5 cm² of the axillary skin correspondingto the area containing sweat pores. The regulated SNARE inhibitor maybetween 1 to 100 units botulinum type A, and preferably 10-50 units, andmost preferably 30 units; or between 1 and 4000 units botulinum B,preferably 20-2000 units, and most preferably 1000 units; or 1-4000units tetanus neurotoxin, preferably 10-1000 units, and most preferably500 units; or between 1 and 1000 units of light chain from botulinumtoxin A, B, C, D, E, F, or G or tetanus toxin, preferable between 10 and500 units and most preferably between 50 and 200 units). An 5 cm² squareelectrode with an oxidized iridium surface is placed over the axillaryskin corresponding to the area containing sweat glands, a distant secondelectrode of between 1 and 20 cm² of surface area is placed on theoutside of the ipsilateral arm. Direct current is passed between theelectrodes with the axillary electrode being the anode. Current rangesfrom 0.1 to 10 mAmp, preferably 10-500 mAmp, most preferably 20-100mAmp. Current is applied for 10 seconds to 20 minutes, preferably 1minute to 10 minutes, most preferably 3-6 minutes. While not wanting tobe bound by theory the regulated SNARE inhibitors diffuse down the sweatducts and are made hydrophilic upon production of an acidic pH producedby the direct current flow. The light chains are then translocated intothe sweat gland cells and their surrounding nerve supply.

In another embodiment of this invention the same patient is treated witha regulated SNARE inhibitor encapsulated into liposomes. A 1 cc solutionof liposomes is placed onto the 5 cm² axillary skin containing the sweatpores. The liposomes contain 1-5000 units of IgA protease, preferably 50to 3000 units, most preferably 500-1000 units; or 1-1000 units ofbotulinum A light chain, preferably 10-500 units, or more preferably100-200 units.

In another embodiment of this invention, the same patient is treatedwith a mixture of regulated SNARE inhibitor and a protein transductiondomain.

8.11 Disease Associated With Holocrine Secretions

In one embodiment of the invention, therapeutically effective amounts ofregulated SNARE inhibitors are administered locally, preferably, by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors—so as to increase or facilitate cell membranetranslocation and/or binding—to treat, reduce the symptoms of, and/orprevent disease associated with holocrine secretions and acne.

Holocrine glands produce sebum. All hair follicles have sebum producingglands that empty into the area of the hair shaft. Excessive productionof sebum can lead to infections of the follicle resulting in a pimple orfuruncle. Sebum is at least partly under the control of substance Psecreting nerves and blocking these nerves decreases sebum production.

8.11 Disease Related To Mucus Secretion

In another embodiment of the invention, therapeutically effectiveamounts of regulated SNARE inhibitors are administered locally,preferably, by way of acid mediated translocation, protein transductiondomains, or encapsulation vectors—so as to increase or facilitate cellmembrane translocation and/or binding—to treat, reduce the symptoms of,and/or prevent disease related to mucous secretion.

Excessive mucous secretion is one of the symptoms of asthma, COPD andcystic fibrosis, and rhinitis in the nose and mucoid otitis media in theear. Acid mediated translocation allows control over these processesindirectly by their innervation and directly by translocation intosecreting cells.

In one example of this embodiment of the invention, a forty-year-oldfemale complains of excessive sweating and odor from her axilla. A warmmoist towel is placed over her axilla to open the sweat pores for tenminutes. Then 1 cc of a normal saline solution containing a regulatedSNARE inhibitor is placed over 5 cm² of the axillary skin correspondingto the area containing sweat pores. The regulated SNARE inhibitor can bebetween 1 to 100 units of botulinum type A, and preferably 10-50 units,and most preferably 30 units; or between 1 and 4000 units botulinum B,preferably 20-2000 units, and most preferably 1000 units; or 1-4000units tetanus neurotoxin, preferably 10-1000 units, and most preferably500 units; or between 1 and 1000 units of light chain from botulinumtoxin A, B, C, D, E, F, or G or tetanus toxin, preferable between 10 and500 units and most preferably between 50 and 200 units). An 5 cm² squareelectrode with an oxidized iridium surface is placed over the axillaryskin corresponding to the area containing sweat glands, a distant secondelectrode of between 1 and 20 cm² of surface area is placed on theoutside of the ipsilateral arm. Direct current is passed between theelectrodes with the axillary electrode being the anode. Current rangesfrom 0.1 to 10 mAmp, preferably 10-500 mAmp, most preferably 20-100mAmp. Current is applied for 10 seconds to 20 minutes, preferably 1minute to 10 minutes, most preferably 3-6 minutes. While not wanting tobe bound by theory the regulated SNARE inhibitors diffuse down the sweatducts and are made hydrophilic upon production of an acidic pH producedby the direct current flow. The light chains are then translocated intothe sweat gland cells and their surrounding nerve supply.

8.12 Prostatic Hypertrophy

In one embodiment of the invention, therapeutically effective amounts ofregulated SNARE inhibitors are administered locally, preferably, by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors—so as to increase or facilitate cell membranetranslocation and/or binding—to treat, reduce the symptoms of, and/orprevent prostatic hypertrophy.

Prostatic hypertrophy is frequent in males over fifty and causesdifficulty urinating due to partial obstruction to urinary flow.Denervation is known to cause involution of the glandular elements ofthe prostate and shrink the gland. Botulinum neurotoxin A has been shownto shrink the gland in rodents.

In one example of this embodiment, a 25-gauge needle is inserted throughthe perineum of a 60-year-old male with prostatic hypertrophy underultrasound guidance. In succession the following solutions are injected,5 ml 1% lidocaine with epinephrine, then 5 ml of 40 units of botulinumneurotoxin A in saline, then 5 ml of acidic solution. The botulinumneurotoxin light chains are translocated into the glandular elements aswell as the innervating neurons. Involution of the gland is noted by oneweek and the patient is free of obstruction for six months.

8.13 Disease Treatable By Gene Therapy

In still one more embodiment of the invention, therapeutically effectiveamounts of regulated SNARE inhibitors are administered locally,preferably, by way of acid mediated translocation, protein transductiondomains, or encapsulation vectors—so as to increase or facilitate cellmembrane translocation and/or binding—to treat, reduce the symptoms of,and/or prevent disease that are treatable by gene therapy.

An amphipathic protein with cargo molecule coding for the membranetransporter of cystic fibrosis is injected via a bronchoscope to onelobe of the lung followed by injection of an acidic solution of pH 4.5.The gene enters the secretory cells and the mucus consistency decreasesto normal. The procedure is repeated every month for each lobe until thedisease is controlled.

8.14 Disease Of The Veins: Venous Stasis, Varicose Veins, Hemorrhoids

In still yet another embodiment of the invention, therapeuticallyeffective amounts of regulated SNARE inhibitors are administered locallyto treat, reduce the symptoms of, and/or prevent disease of the veins,such as venous stasis, varicose veins, and hemorrhoids.

Veins are high compliance low-pressure vessels. With aging, they becomeweaker resulting in dilatation manifest as varicose veins in thesubcutaneous tissue and hemorrhoids around the anal sphincter. Deeperveins can allow blood to stagnate and clot causing deep venousthrombosis and pulmonary emboli. Vein contraction is adrenergictherefore increasing the sympathetic tone causes contraction andrelieves many of these conditions. In addition, sympathetic nerves areunregulated with increasing NGF.

In one example of this embodiment of the invention, a 60-year-old femalesuffers from varicose veins on the back of her hands that arecosmetically undesirable. About 5 units of tetanus neurotoxin in a 5 mlsolution is administered by percutaneous injection around the veins. Aweek later the tetanus neurotoxin has been transported retrograde andcauses increased sympathetic tone to the veins. They in turn constrictand become smaller and less noticeable.

In another example of this embodiment, a fifty-year-old female hasvaricose veins in her legs. She has 5 IU of tetanus toxoid injectedaround the veins. The sympathetic neurons increase in size and thevaricose veins shrink.

In another example, a 60-year-old male with hemorrhoids is treated byadministering an anal suppository containing 5 units of tetanusneurotoxin in a rapid degradable gelatin. After an hour he expels thesuppository. In one week the sympathetic tone to the hemorrhoids hasincreased and they shrink.

In still another example, a solution containing a hybrid tetanusneurotoxin suspended in normal saline is administered to the nose of athirty-year-old male suffering from a viral URI (cold) with nasalcongestion. The tetanus neurotoxin has had its light chain replaced bythat from botulinum neurotoxin E, whose duration of action is one day.The next day the sympathetic activity to the venous cavities of thenasal mucosa increases and their size shrinks thereby relieving hisnasal congestion for a day. Application of botulinum neurotoxin E couldalso be done to decrease rhinorrhea and neurogenic inflammation.

In yet another example, a combination of botulinum neurotoxin, acholinergic blocker, with tetanus neurotoxin, a cholinergic (that isblocked) and adrenergic agonist is administered for beneficial effectgreater then can be achieved by each alone. The parasympathetic andsympathetic nervous system often innervate and have opposite effects onthe same organ.

8.15 High Blood Pressure

In still yet another embodiment of the invention of the invention,therapeutically effective amounts of regulated SNARE inhibitors areadministered locally to treat, reduce the symptoms of, and/or preventhigh blood pressure.

In animals, injection of tetanus neurotoxin into the anterior stomachwall produces decreased blood pressure. Presumably this is due to itstransport through the vagus to the nucleus ambiguus where it can diffuseto the neighboring external formation where cardiovascular controlneurons are present. This is another example of using the circuitry ofthe CNS to effect a target indirectly.

In an example of this embodiment of the invention, a fifty-year-oldblack male with severe hypertension undergoes fiberoptic laparoscopywhere a 1 week biodegradable pellet containing 5 units of tetanusneurotoxin is injected into the anterior stomach wall. The tetanusneurotoxin is transported retrograde to the brainstem and the patient'sblood pressure decreases for three months.

8.16 Use Regulated SNARE Inhibitors Simultaneously as a TherapeuticAgent and as a Vaccine

In another embodiment of the invention, therapeutically effectiveamounts of regulated SNARE inhibitors are administered locally,preferably, by way of acid mediated translocation, protein transductiondomains, or encapsulation vectors—so as to increase or facilitate cellmembrane translocation and/or binding—to act simultaneously as atherapeutic agent and as a Vaccine.

Most humans and farm animals are vaccinated against tetanus neurotoxin,in fact, the vaccine is the largest biopharmaceutical product in theworld. Instead of separate vaccinations of toxoid and therapeuticapplications of tetanus neurotoxin, tetanus neurotoxin can be used forboth. Especially in farm animals a single injection of slow releasewould have the therapeutic effect while still vaccinating the animal.

In an example of this embodiment of the invention, 200 units of tetanusneurotoxin in a 2 month biodegradable microparticle is injected into thetail of a cow. The tetanus neurotoxin is slowly released and istransported retrograde. As the amount is in excess of what theprojection neurons from the tail can absorb, the remainder diffuses intothe CSF or is transported retrograde to cause general motordisinhibition, that is widespread increases in muscle tone to increasemeat production, while simultaneous causing vaccination. This examplealso demonstrates that tetanus neurotoxin can be used to causedisinhibition in second or third order projection neurons.

CONCLUSION

From the above summary, description and examples, it is clear that incertain embodiments, the invention relates to:

In one embodiment, the invention relates to a methods and compositionsfor modulating cellular function in a mammal comprising locallyadministering a modulatorily effective amount of a regulated SNAREinhibitor and a translocating agent to the mammal, whereby translocationof the regulated SNARE inhibitor is facilitated.

In another embodiment, the invention relates to methods and compositionsfor modulating cellular function in a mammal comprising locallyadministering an modulatorily effective amount of a regulated SNAREinhibitor to the mammal at a selected site and decreasing a pH value atthe selected site.

In yet another embodiment, the invention relates to methods andcompositions for modulating cellular function in a mammal comprisinglocally administering an modulatorily effective amount of a regulatedSNARE inhibitor to the mammal at a selected site, by way of a proteintransduction domain.

In still another embodiment, the invention relates to methods andcompositions for modulating cellular function in a mammal comprisinglocally administering an modulatorily effective amount of a regulatedSNARE inhibitor to the mammal at a selected site, by way of anencapsulation vector.

In one more embodiment, the invention relates to methods andcompositions for modulating cellular function of a mammal suffering froma disease or medical condition, whereby the disease or medical conditionis treated.

In still yet another embodiment, the invention relates to methods andcompositions for modulating cellular function of a mammal suffering froma disease or medical condition comprising locally administering atherapeutically effective amount of a regulated SNARE inhibitor to amammal in need of such treatment at a selected site and decreasing a pHvalue at the selected site.

In another embodiment, the invention relates to methods and compositionsfor modulating cellular function of a mammal suffering from a disease,malfunction, or dysfunction comprising locally administering atherapeutically effective amount of a regulated SNARE inhibitor to amammal in need of such treatment at a selected site by way of a proteintransduction domain.

In a further embodiment, the invention relates to methods andcompositions modulating cellular function of a mammal suffering from adisease, malfunction, or dysfunction comprising locally administering atherapeutically effective amount of a regulated SNARE inhibitor to amammal in need of such treatment at a selected site by way of anencapsulation vector.

In another embodiment, the invention relates to pharmaceuticalformulations for modulating cellular function in a mammal comprising atherapeutically effective amount of a regulated SNARE inhibitor and atranslocating agent in a pharmaceutically acceptable carrier for localdelivery. Preferably, the translocating agent is an acid, an acidicenvironment, an encapsulating vector, or a transduction domain and theregulated SNARE inhibitor is a bacterial neurotoxin.

In yet a further embodiment, the invention relates to the use of aregulated SNARE inhibitors in the preparation of a medicament for amethod of treating a disease or medical condition in a mammal comprisinglocally administering a therapeutically effective amount of a regulatedSNARE inhibitor to a mammal in need of such treatment at a selected siterelated to the disease, by way of acid mediated translocation, a proteintransduction domain, or an encapsulation vector.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples, which are intended asillustrations of a few aspects of the invention. Any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.All cited references are hereby incorporated herein in their entiretiesby reference.

1.
 116. (canceled)
 117. A method of treating or reduction of symptomsassociated with inflammation in a mammal in need thereof comprisingadministering a therapeutically effective amount of a botulinumneurotoxin to treat or reduce symptoms associated with inflammation intothe mammal's pterygopalatine space, thereby blocking the mammal'ssphenopalatine ganglion.
 118. The method of claim 117, wherein thebotulinum neurotoxin is administered by injection.
 119. The method ofclaim 117, wherein the botulinum neurotoxin is topically administered.120. The method of claim 117, wherein the botulinum neurotoxin isselected from the group consisting of botulinum neurotoxin serotypes A,B, C1, D, E, F and G.
 121. The method of claim 119, wherein thebotulinum neurotoxin is botulinum neurotoxin serotype A.
 122. The methodof claim 117, wherein said administering step comprises the steps ofpassing a needle about 1.5 inches through the sphenopalatine canal andinjecting the botulinum neurotoxin into the mammal's pterygopalatinespace.
 123. The method of claim 117, wherein said symptoms associatedwith inflammation includes congestion, primary inflammation andneurogenic inflammation.
 124. The method of claim 117, wherein saidadministration of a therapeutically effective amount of botulinumneurotoxin decreases rhinorrhea, congestion, primary inflammation,neurogenic inflammation, or combinations thereof.
 125. The method ofclaim 117, wherein said administration of a therapeutically effectiveamount of botulinum neurotoxin inhibits mast cell release of bioactivesubstances.
 126. The method of claim 117, wherein the botulinumneurotoxin is administered at a dosage of between about 0.1 units toabout 1000 units.
 127. The method of claim 117, wherein the botulinumneurotoxin is administered at a dosage of between about 1 unit to about100 units.
 128. The method of claim 117, wherein the botulinumneurotoxin is administered by way of acid mediated translocation,protein transduction domains, or encapsulation vectors.
 129. A method oftreating or reduction of symptoms associated with inflammation in amammal in need thereof comprising administered a therapeuticallyeffective amount of botulinum neurotoxin serotype A to treat or reducesymptoms associated with inflammation into the mammal's pterygopalatinespace, thereby blocking the mammal's sphenopalatine ganglion.
 130. Themethod of claim 129, wherein said symptoms associated with inflammationincludes congestion, primary inflammation and neurogenic inflammation.131. The method of claim 129, wherein said administration of atherapeutically effective amount of botulinum neurotoxin serotype Adecreases rhinorrhea, congestion, primary inflammation and neurogenicinflammation.
 132. The method of claim 129, wherein said administrationof a therapeutically effective amount of botulinum neurotoxin serotype Ainhibits mast cell release of bioactive substances.
 133. The method ofclaim 129, wherein said administering step comprises the steps ofpassing a needle about 1.5 inches through the sphenopalatine canal andinjecting the botulinum neurotoxin serotype A into the mammal'spterygopalatine space.
 134. The method of claim 129, wherein thebotulinum neurotoxin serotype A is administered at a dosage of betweenabout 0.1 units to about 1000 units.
 135. The method of claim 129,wherein the botulinum neurotoxin serotype A is administered at a dosageof between about 1 unit to about 100 units.
 136. The method of claim129, wherein the botulinum neurotoxin serotype A is administered by wayof acid mediated translocation, protein transduction domains, orencapsulation vectors.